Antibodies that neutralize botulinum neurotoxins

ABSTRACT

This disclosure provides antibodies that specifically bind to and typically neutralize botulinum neurotoxins (e.g., BoNT/A, BoNT/B, BoNT/E, etc.) and the epitopes bound by those antibodies. The antibodies and derivatives thereof and/or other antibodies that specifically bind to the neutralizing epitopes provided herein can be used to neutralize botulinum neurotoxin and are therefore also useful in the treatment of botulism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. provisional applicationSer. No. 61/085,328 filed on Jul. 31, 2008, which application isincorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant No.DAMD17-98-C-8030, awarded by the United States Army Medical Research andMateriel Command, and grant No. U01 AI056493, awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

This disclosure relates antibodies that neutralize botulinum neurotoxins(e.g., BoNT/A) and their use in the treatment of botulism.

INTRODUCTION

Botulism is caused by botulinum neurotoxin secreted by members of thegenus Clostridium and is characterized by flaccid paralysis, which ifnot immediately fatal requires prolonged hospitalization in an intensivecare unit and mechanical ventilation. Naturally occurring botulism isfound in infants or adults whose gastrointestinal tracts becomecolonized by Clostridial bacteria (infant or intestinal botulism), afteringestion of contaminated food products (food botulism), or in anaerobicwound infections (wound botulism) (Center for Disease Control (1998)Botulism in the United States, 1899-1998. Handbook for epidemiologists,clinicians, and laboratory workers. Atlanta, Ga. U.S. Department ofHealth and Human Services, Public Health Service: downloadable at“bt.cdc.gov/agent/botulism/index.asp”). Botulism neurotoxins (BoNTs) arealso classified by the Centers for Disease Control (CDC) as one of thesix highest-risk threat agents for bioterrorism (the “Category Aagents”), due to their extreme potency and lethality, ease of productionand transport, and need for prolonged intensive care (Amon et al. (2001)JAMA 285: 1059-1070). As a result of these threats, specificpharmaceutical agents are needed for prevention and treatment ofintoxication.

No specific small molecule drugs exist for prevention or treatment ofbotulism, but an investigational pentavalent toxoid vaccine is availablefrom the CDC (Siegel (1988) J. Clin. Microbiol. 26: 2351-2356) and arecombinant vaccine is under development (Smith (1998) Toxicon 36:1539-1548). Regardless, mass civilian or military vaccination isunlikely due to the rarity of disease or exposure and the fact thatvaccination would prevent subsequent medicinal use of BoNT.Post-exposure vaccination is useless, due to the rapid onset of disease.Toxin neutralizing antibody (Ab) can be used for pre- or post-exposureprophylaxis or for treatment (Franz et al. (1993) Pp. 473-476 In B. R.DasGupta (ed.), Botulinum and Tetanus Neurotoxins: Neurotransmission andBiomedical Aspects. Plenum Press, New York). Small quantities of bothequine antitoxin and human botulinum immune globulin exist and arecurrently used to treat adult (Black and Gunn. (1980) Am. J. Med., 69:567-570; Hibbs et al. (1996) Clin. Infect. Dis., 23: 337-340) and infantbotulism (Amon (1993). Clinical trial of human botulism immune globulin,p. 477-482. In B. R. DasGupta (ed.), Botulinum and Tetanus Neurotoxins:Neurotransmission and Biomedical Aspects. Plenum Press, New York)respectively.

The development of mAb therapy for botulism is complicated by the factthat there are at least seven BoNT serotypes (A-G) (Hatheway (1995)Curr. Top. Microbio. Immunol, 195: 55-75) that show little, if any,antibody cross-reactivity. While only four of the BoNT serotypesroutinely cause human disease (A, B, E, and F), there has been onereported case of infant botulism caused by BoNT C (Oguma et al. (1990)Lancet 336: 1449-1450), one outbreak of foodborne botulism linked toBoNT D (Demarchi, et al. (1958) Bull. Acad. Nat. Med., 142: 580-582),and several cases of suspicious deaths where BoNT G was isolated(Sonnabend et al. (1981) J. Infect. Dis., 143: 22-27). AerosolizedBoNT/C, D, and G have also been shown to produce botulism in primates bythe inhalation route (Middlebrook and Franz (1997) Botulinum Toxins,chapter 33. In F. R. Sidell, E. T. Takafuji, D. R. Franz (eds.), MedicalAspects of Chemical and Biological Warfare. TMM publications,Washington, D.C.), and would most likely also affect humans. Thus, it islikely that any one of the seven BoNT serotypes can be used as abiothreat agent.

Variability of the BoNT gene and protein sequence within serotypes hasalso been reported and there is evidence that such variability canaffect the binding of monoclonal antibodies to BoNT/A (Kozaki et al.(1998) Infect. Immun., 66: 4811-4816; Kozaki et al. (1995) Microbiol.Immunol., 39: 767-774).

SUMMARY

Antibodies that bind to and neutralize and/or otherwise clear botulinumneurotoxin(s) are disclosed herein. Particularly effectiveneutralization of a Botulism neurotoxin (BoNT) serotype can be achievedby the use of neutralizing antibodies that bind two or more subtypes ofthe particular neurotoxin serotype with particularly high affinityand/or by combinations of such antibodies. The present disclosureprovides antibodies that bind BoNT subtypes BoNT/A, BoNT/B, and BoNT/E.Compositions comprising neutralizing antibodies that bind two or moreBoNT subtypes (e.g., BoNT/B1, BoNT/B2, BoNT/B3, etc.) with high affinityare also provided herein.

A neutralizing antibody for Botulinum neurotoxin (BoNT) is providedherein. The antibody typically comprises at least one VH complementaritydetermining region (CDR) selected from the group consisting of a 2A10 VHCDR, a 3E1VH CDR, a 3E2VH CDR, a 3E3VH CDR, a 3E4VH CDR, a 3E4.1VH CDR,a 3E5VH CDR, a 3E6VH CDR, a 3E6.1VH CDR, a 4E1 IVH CDR, a 4E13VH CDR, a4E16VH CDR, a 4E16.1VH CDR, a 4E17VH CDR, a 4E17.1VH CDR, an A12 VH CDR,a 6A12 VH CDR, a B1.1 VH CDR, a B6 VH CDR, a B6.1 VH CDR, a B8 VH CDR, aB8.1 VHCDR, a B11 VH CDR, a B11C3 VH CDR, a B11E8 VH CDR, a B12 VH CDR,a B12.1 VH CDR, a B12.2 VH CDR, a 1B18 VH CDR, a 2B18.1 VH CDR, a 4B19VH CDR, and a 1B22 VH CDR, a 1B10 VH CDR, a 1B10.1 VH CDR, a 2B18.2 VHCDR, a 5 2B18.3 VH CDR, a 1B22.4 VH CDR, a 2B23 VH CDR, a 2B24 VH CDR, a2B25 VH CDR, a 2B25.1 VH CDR, a 2B26 VH CDR, a 2B27 VH CDR, a 2B28 VHCDR, a 2B29 VH CDR, a 2B30 VH CDR, a 4B17.1 VH CDR, a 4B17.1C VH CDR, a4B17.1D VH CDR, a 4B17.1F VH CDR, a 4B17.1G VH CDR, a 3E6.2 VH CDR, a4E17.4 VH CDR, a 4E17.6 VH CDR, B11.H12 VH CDR, B11.E9 VH CDR, 4B1 VHCDR, 4B3 VH CDR, 4B5 VH CDR, 4B6 VH CDR, 4B7 VH CDR, 1B14 VH CDR, 4A1 VHCDR, 4A1.1 VH CDR, 5A20.4 VH CDR, ING1.1C1 VH CDR, ING1.5B1 VH CDR,ING1.2B10 VH CDR, and ING1.3C2 VH CDR, and/or at least one VLcomplementarity determining region selected from the group consisting ofa 2A10 VL CDR, a 3E1VL CDR, a 3E2 VL CDR, a 3E3 VL CDR, a 3E4 VL CDR, a3E4.1VL CDR, a 3E5 VL CDR, a 3E6 VL CDR, a 3E6.1 VL CDR, a 4E11 VL CDR,a 4E13 VL CDR, a 4E16 VL CDR, a 4E16.1 VL CDR, a 4E17 VL CDR, a 4E17.1VL CDR, an A12 VL CDR, a 6A12 VL CDR, a B1.1 VL CDR, a B6 VL CDR, a B6.1VL CDR, a B8 VL CDR, a B8.1 VL CDR, a B11 VLCDR, a B11C3 VL CDR, a B11E8VL CDR, a B12 VL CDR, a B12.1 VL CDR, a B12.2 VL CDR, a 1B18 VL CDR, a2B18.1 VL CDR, a 4B19 VL CDR, and a 1B22 VL CDR, a 1B10 VL CDR, a 1B10.1VL CDR, a 2B18.2 VL CDR, a 2B18.3 VL CDR, a 1B22.4 VL CDR, a 2B23 VLCDR, a 2B24 VL CDR, a 2B25 VL CDR, a 2B25.1 VL CDR, a 2B26 VL CDR, a2B27 VL CDR, a 2B28 VL CDR, a 2B29 VL CDR, a 2B30 VL CDR, a 4B17.1 VLCDR, a 4B17.1C VL CDR, a 4B17.1D VL CDR, a 4B17.1F VL CDR, a 4B17.1G VLCDR, a 3E6.2 VL CDR, a 4E17.4 VL CDR, a 4E17.6 VL CDR, B11.H12 VL CDR,B11.E9 VL CDR, 4B1 VL CDR, 4B3 VL CDR, 4B5 VL CDR, 4B6 VL CDR, 4B7 VLCDR, 1B14 VL CDR, 4A1 VL CDR, 4A1.1 VL CDR, 5A20.4 VL CDR, ING1.1C1 VLCDR, ING1.5B1 VL CDR, ING1.2B10 VL CDR, and ING1.3C2 VL CDR.

The antibody may contain the VH CDRs of an antibody selected from thegroup consisting of 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23, 2B24,2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C, 4B17.1D,4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6 B11.H12, B11.E9, 4B1, 4B3, 4B5,4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, andING1.3C2; and/or the VL CDRs of an antibody selected from the groupconsisting of 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23, 2B24, 2B25,2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C, 4B17.1D, 4B17.1F,4B17.1G, 3E6.2, 4E17.4, 4E17.6 B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7,1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2.

The antibody may contain the VH and VL CDRs of an antibody selected fromthe group consisting of 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23,2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6 B11.H12, B11.E9, 4B1,4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1,ING1.2B10, and ING1.3C2

The antibody may contain the VH and VL domains of an antibody selectedfrom the group consisting of 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23,2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6 B11.H12, B11.E9, 4B1,4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1,ING1.2B10, and ING1.3C2.

The antibody may be a single chain Fv (scFv), a Fab, a (Fab′)₂, an(ScFv)₂, and the like. The antibody may be an IgG. The antibody may beselected from the group consisting of 1B10, 1B10.1, 2B18.2, 2B18.3,1B22.4, 2B23, 2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1,4B17.1C, 4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, B6.1, B11E8, 4E17.1, 4E16.1,3E6.1, B12.1, 4E17.4, 4E17.6 B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7,1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2.The antibody may also be in a pharmaceutically acceptable excipient(e.g., in a unit dosage formulation).

Methods of inhibiting the activity of Botulinum neurotoxin in a mammalmay involve administering to a mammal in need thereof a compositioncomprising at least one neutralizing anti-BoNT antibody as describedherein. The composition may include at least two different antibodies,each of which binds to different BoNT subtypes. The composition may alsoinclude at least three, at least four, or more different antibodies,each of which may bind to different BoNT epitopes.

Compositions provided herein may partially or fully neutralize aBotulinum neurotoxin (BoNT). The compositions typically include a firstantibody that binds a BoNT/B or a BoNT/E serotype, e.g, one or moreantibodies as described above, and a second antibody that binds a BoNTserotype selected from the group consisting of BoNT/A, BoNT/B, BoNT/C,BoNT/D, BoNT/E, and BoNT/F.

Nucleic acids provided herein encode one or more antibodies that aredescribed herein. Cells containing such antibodies are also providedherein. Kits provided for neutralizing a Botulinum neurotoxin mayinclude a composition containing one or more antibodies as describedherein. The kits optionally also include instructional materialsteaching the use of the composition to neutralize a Botulinumneurotoxin. The composition may be stored in a disposable syringe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows deduced protein sequences of heavy (VH) and light (VL)chain variable regions of BoNT B binders. VH domains: A12 (SEQ ID NO:1),6A12 (SEQ ID NO:2), B1.1 (SEQ ID NO:3), B6 (SEQ ID NO:4), B6.1 (SEQ IDNO:5), B8 (SEQ ID NO:6), B8.1 (SEQ ID NO:7), B11 (SEQ ID NO:8), B11C3(SEQ ID NO:9), B11E8 (SEQ ID NO:10), B12 (SEQ ID NO:11), B12.1 (SEQ IDNO:12), B12.2 (SEQ ID NO:13), 1B18 (SEQ ID NO:14), 2B18.1 (SEQ IDNO:15), 4B19 (SEQ ID NO:16), 1B22 (SEQ ID NO:17), 1B10 (SEQ ID NO:18),1B10.1 (SEQ ID NO:19), 2B18.2 (SEQ ID NO:20), 2B18.3 (SEQ ID NO:21),1B22.4 (SEQ ID NO:22), 2B23 (SEQ ID NO:23), 2B24 (SEQ ID NO:24), 2B25(SEQ ID NO:25), 2B25.1 (SEQ ID NO:26), 2B26 (SEQ ID NO:27), 2B27 (SEQ IDNO:28), 2B28 (SEQ ID NO:29), 2B29 (SEQ ID NO:30), 2B30 (SEQ ID NO:31),4B17.1 (SEQ ID NO:32), 4B17.1C (SEQ ID NO:33), 4B17.1D (SEQ ID NO:34),4B17.1F (SEQ ID NO:35), 4B17.1G (SEQ ID NO:36). VL domains: A12 (SEQ IDNO:37), 6A12 (SEQ ID NO:38), B1.1 (SEQ ID NO:39), B6 (SEQ ID NO:40),B6.1 (SEQ ID NO:41), B8 (SEQ ID NO:42), B8.1 (SEQ ID NO:43), B11 (SEQ IDNO:44), B11C3 (SEQ ID NO:45), B11E8 (SEQ ID NO:46), B12 (SEQ ID NO:47),B12.1 (SEQ ID NO:48), B12.2 (SEQ ID NO:49), 1B18 (SEQ ID NO:50), 2B18.1(SEQ ID NO:51), 4B19 (SEQ ID NO:52), 1B22 (SEQ ID NO:53), 1B10 (SEQ IDNO:54), 1B10.1 (SEQ ID NO:55), 2B18.2 (SEQ ID NO:56), 2B18.3 (SEQ IDNO:57), 1B22.4 (SEQ ID NO:58), 2B23 (SEQ ID NO:59), 2B24 (SEQ ID NO:60),2B25 (SEQ ID NO:61), 2B25.1 (SEQ ID NO:62), 2B26 (SEQ ID NO:63), 2B27(SEQ ID NO:64), 2B28 (SEQ ID NO:65), 2B29 (SEQ ID NO:66), 2B30 (SEQ IDNO:67), 4B17.1 (SEQ ID NO:68), 4B17.1C (SEQ ID NO:69), 4B17.1D (SEQ IDNO:70), 4B17.1F (SEQ ID NO:71), 4B17.1G (SEQ ID NO:72).

FIG. 2 shows deduced protein sequences of heavy and light chain variableregions of BoNT/E binders. VH domains: 2A10 (SEQ ID NO:73), 3E1 (SEQ IDNO:74), 3E2 (SEQ ID NO:75), 3E3 (SEQ ID NO:76), 3E4 (SEQ ID NO:77),3E4.1 (SEQ ID NO:78), 3E5 (SEQ ID NO:79), 3E6 (SEQ ID NO:80), 3E6.1 (SEQID NO:81), 4E11 (SEQ ID NO:82), 4E13 (SEQ ID NO:83), 4E16 (SEQ IDNO:84), 4E16.1 (SEQ ID NO:85), 4E17 (SEQ ID NO:86), 4E17.1 (SEQ IDNO:87), 3E6.2 (SEQ ID NO:88), 4E17.4 (SEQ ID NO:89), 4E17.6 (SEQ IDNO:90); VL domains: 2A10 (SEQ ID NO:91), 3E1 (SEQ ID NO:92), 3E2 (SEQ IDNO:93), 3E3 (SEQ ID NO:94), 3E4 (SEQ ID NO:95), 3E4.1 (SEQ ID NO:96),3E5 (SEQ ID NO:97), 3E6 (SEQ ID NO:98), 3E6.1 (SEQ ID NO:99), 4E11 (SEQID NO:100), 4E13 (SEQ ID NO:101), 4E16 (SEQ ID NO:102), 4E16.1 (SEQ IDNO:103), 4E17 (SEQ ID NO:104), 4E17.1 (SEQ ID NO:105), 3E6.2 (SEQ IDNO:106), 4E17.4 (SEQ ID NO:107), 4E17.6 (SEQ ID NO:108).

FIG. 3 shows a phylogenetic tree of published botulinum neurotoxingenes. The phylogenetic tree was constructed from the DNA sequences ofpublished Clostridial neurotoxin genes using Vector NTI software.

FIG. 4 shows an analysis of BoNT/B gene sequences. A phylogenetic treeof BoNT/B genes reveals four clusters: BoNT/B1, BoNT/B2, nonproteolyticBoNT/B, and bivalent BoNT/B. Percent differences between clusters rangefrom 3.6 to 7.7%. As with BoNT/A, the greatest differences are seen inthe heavy chain.

FIGS. 5A and 5B show a scheme used for affinity maturation of HuC25(FIG. 5A) and 3D12 (FIG. 5B) scFv using yeast display.

FIG. 6 illustrates mapping toxin domains recognized by mAbs by usingyeast displayed BoNT domains. BoNT/B binding domain Hc), translocationdomain (H_(N)), and light chain (L_(C)) were well displayed on the yeastsurface as evidenced by staining with anti-SV5-ALEXA FLUOR 647 dye (APCchannel). Each domain was only bound by a mAb specific for 20 thatdomain, but not by other BoNT/B mAbs as evidenced by staining with mAb6.1 (L_(C) specific), mAb 1B 12.1 (H_(C) specific), and mAb 1B18 (H_(N)specific) detected with anti-human phycoerythrin (PE channel).

FIG. 7 shows a model of the epitopes of BoNT/B mAbs. The data generatedby the studies described in FIG. 6 were used to map each scFv to theBoNT/B domain that it bound. scFv binding overlapping epitopes areindicated by overlap of the circle representing the scFv.Non-overlapping circles indicate that the scFv epitopes do not overlap.Otherwise, the scFv epitopes have been randomly placed on each of theBoNT/B domains. Modeling was done on the X-ray crystal structure ofBoNT/B.

FIG. 8 illustrates the selection of yeast displayed antibodies crossreactive with BoNT/A subtypes. Dot-plots of flow cytometry sorting ofscFv displaying yeast labeled with BoNT/A are shown. For each of thefour rounds of sorting, the concentration of BoNT/A1 or BoNT/A2 used tostain yeast is indicated. For the first two rounds of sorting, BoNT/Abinding is indicated on the Y-axis and the scFv display level on theX-axis. For the last two rounds of sorting, scFv displaying yeast whichbound toxin epitopes that did not overlap with mAb AR1 or mAb 3D12 areindicated on the Y and X axes respectively (see methods section forstaining details). The sort gates used for yeast collection areindicated and the yeast in these gates are colored green.

FIG. 9 shows a schematic of one method used to construct yeast displayedlight chain shuffled scFv antibody libraries. A. V_(L) gene repertoireswere amplified by using PCR from donor cDNA or cloned scFv generepertoires. B. V_(L) gene repertoires are reamplified to append theV_(H) framework 4, scFv linker, and cloning sites at the 5′ and 3′ endof the genes. C. The V_(L) gene repertoire is cloned into the yeastdisplay vector pYD2. D. The V_(H) gene of a binding scFv is PCRamplified using primers that append overhangs complementary to NcoI-NotIor HindIII-NotI digested pYD2-V_(L) gene repertoire vector DNA. E. TheV_(H) gene is cloned into the pYD-V_(L) repertoire vector to create alight chain shuffled library. Gal1-10 promoter=galactose promoter; synprepro leader=synthetic leader sequence; Aga1=Aga1 surface protein;Aga2=Aga2 surface protein gene; trp=tryptophan selectable marker.

FIG. 10 is a schematic showing the relationship of certain antibodiesspecific for BoNT/E and BoNT/B disclosed herein with respect to theclonal lineage of the first lead selected.

FIG. 11 is a schematic providing the amino acid sequences of the V_(H)and V_(L) of certain antibodies disclosed herein. Panels A and B presentV_(H) and V_(L) amino acid sequences, respectively, for BoNT/A bindingantibodies, while panels C and D present V_(H) and V_(L) amino acidsequences, respectively, for BoNT/B binding antibodies.

FIG. 11, Panel A, V_(H) domains: ING1 (SEQ ID NO:939), ING1.1C1 (SEQ IDNO:940), ING1.2B10 (SEQ ID NO:941), ING1.5B1 (SEQ ID NO:942), ING1.3C2(SEQ ID NO:943), 4A1 (SEQ ID NO:944), 4A1.1 (SEQ ID NO:945), 5A20 (SEQID NO:946).

FIG. 11, Panel B, V_(L) domains: ING1 (SEQ ID NO:947), ING1.1C1 (SEQ IDNO:948), ING1.2B10 (SEQ ID NO:949), ING1.5B1 (SEQ ID NO:950), ING1.3C2(SEQ ID NO:951), 4A1 (SEQ ID NO:952), 4A1.1 (SEQ ID NO:953), 5A20 (SEQID NO:954).

FIG. 11, Panel C, V_(H) domains: 4B1 (SEQ ID NO:955), 4B3 (SEQ IDNO:956), 4B5 (SEQ ID NO:957), 4B6 (SEQ ID NO:958), B6.1 (SEQ ID NO:959),B6.C12 (SEQ ID NO:960), B6.D2 (SEQ ID NO:961), 4B7 (SEQ ID NO:962), B11(SEQ ID NO:963), B11.A5 (SEQ ID NO:964), B11.B9 (SEQ ID NO:965), B11.F7(SEQ ID NO:966), B11.H12 (SEQ ID NO:967), 1B14 (SEQ ID NO:968), 4B19.1(SEQ ID NO:969).

FIG. 11, Panel D, V_(L) domains: 4B1 (SEQ ID NO:970), 4B3 (SEQ IDNO:971), 4B5 (SEQ ID NO:972), 4B6 (SEQ ID NO:973), B6.1 (SEQ ID NO:974),B6.C12 (SEQ ID NO:975), B6.D2 (SEQ ID NO:976), 4B7 (SEQ ID NO:977), B11(SEQ ID NO:978), B11.A5 (SEQ ID NO:979), B11.B9 (SEQ ID NO:980), B11.F7(SEQ ID NO:981), B11.H12 (SEQ ID NO:982), 1B14 (SEQ ID NO:983), 4B19.1(SEQ ID NO:984).

FIG. 12 depicts the amino acid sequences of the V_(H) and V_(L) of1B10.1, in which the bolded amino acid residues are the CDRs accordingto the IMGT definition while the underlined residues represent the CDRsaccording to Kabat et al., supra.

DEFINITIONS

A “BoNT polypeptide” refers to a Botulinum neurotoxin polypeptide (e.g.,a BoNT/A polypeptide, a BoNT/B polypeptide, a BoNT/C polypeptide, and soforth). The BoNT polypeptide can refer to a full-length polypeptide orto a fragment thereof. Thus, for example, the term “BoNT/A polypeptide”refers to either a full-length BoNT/A (a neurotoxin produced byClostridium botulinum of the type A serotype) or a fragment thereof(e.g. the H_(C) fragment). The H_(C) fragment of BoNT/A is anapproximately 50 kDa C-terminal fragment (residues 873-1296) of BoNT/A(Lacy and Stevens (1999) J. Mol. Biol., 291: 1091-1104).

A “BoNT” serotype refers one of the standard known BoNT serotypes (e.g.BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G etc.). BoNTserotypes differ from each other by as little as about 35% at the aminoacid level (e.g., between BoNT/E and BoNT/F) up to about 66% at theamino acid level, (e.g., for BoNT/A vs BoNT/C or D). Thus, BoNTserotypes differ from each other by about 35-66% at the amino acidlevel.

The term “BoNT subtype” (e.g., a BoNT/A1 subtype) refers to botulinumneurotoxin gene sequences of a particular serotype (e.g., A, B, C, D, E,F, etc.) that differ from each other sufficiently to producedifferential antibody binding. The subtypes may differ from each otherby at least 2.5%, by at least 5%, by at least 10%, by at least 15% or upto about at least 20% at the amino acid level. The subtypes differ fromeach other by no more than 35%, by no more than 31.6%, by no more than30%, or 25%, by less than about 20% or 16% at the amino acid level. BoNTsubtypes may differ from each other by at least 2.6%, by at least 3%,and by at least 3.6% at the amino acid level. BoNT subtypes typicallydiffer from each other by less than about 31.6%, by less than about 16%,at the amino acid level, other by less than about 31.6%, by less thanabout 16%, at the amino acid level.

An “anti-BoNT antibody” refers to an antibody that binds a BoNTpolypeptide, specifically binds a BoNT polypeptide with a K_(D) lessthan 10⁻⁷, less than 10⁻⁸, less than 10⁻⁹, less than 10⁻¹⁰, less than10⁻¹¹, or less than 10⁻¹² orless.

“Neutralization” refers to a measurable decrease in the toxicity and/orcirculating level of a Botulinum neurotoxin (e.g., BoNT/A).

“Potency” refers to the degree of protection from challenge with BoNT.This can be measured/quantified for example, as an increase in the LD₅₀of a Botulinum neurotoxin (BoNT). In toxicology, the median lethal dose,LD₅₀ (abbreviation for “Lethal Dose, 50%”), or LCt₅₀ (LethalConcentration & Time) of a toxic substance or radiation is the doserequired to kill half the members of a tested population. The LD₅₀usually expressed as the mass of substance administered per unit mass oftest subject, such as grams of substance per kilogram of body mass.Stating it this way allows the relative toxicity of different substancesto be compared, and normalizes for the variation in the size of theanimals exposed (although toxicity does not always scale simply withbody mass). Typically, the LD₅₀ of a substance is given in milligramsper kilogram of body weight. In the case of some toxins, the LD₅₀ may bemore conveniently expressed as micrograms per kilogram (μg/kg) of bodymass.

The term “high affinity” when used with respect to an antibody refers toan antibody that specifically binds to its target(s) with an affinity(K_(D)) of at least about 10⁻⁸ M, preferably at least about 10⁻⁹ M, atleast about 10⁻¹⁰ M, and at least about 10⁻¹¹ M. “High affinity”antibodies may have a K_(D) that ranges from about 1 nM to about 5 pM.

The following abbreviations are used herein: AMP, ampicillin; BIG,botulinum immune globulin; BoNT, botulinum neurotoxin; BoNT/A, BoNT typeA; BoNT/B: Botulinum neurotoxin serotype B, BoNT/A H_(C): Botulinumneurotoxin serotype A binding domain, C-terminal domain of the BoNT/Aheavy chain; BoNT/A H_(N): Botulinum neurotoxin serotype A translocationdomain, N-terminal domain of the BoNT/A heavy chain; BoNT/A L_(C):Botulinum neurotoxin serotype A catalytic domain; CDR, complementaritydetermining region; ELISA, enzyme-linked immunosorbent assay; GLU,glucose; HBS, HEPES-buffered saline (10 mM HEPES, 150 mM NaCl [pH 7.4]);IgG, immunoglobulin G; IMAC, immobilized-metal affinity chromatography;IPTG, isopropyl-β-D-thiogalactopyranoside; KAN, kanamycin; K_(D),equilibrium constant; k_(off), dissociation rate constant; k_(on),association rate constant; MPBS, skim milk powder in PBS; NTA,nitrilotriacetic acid; PBS, phosphate-buffered saline (25 mM NaH₂PO₄,125 mM NaCl [pH 7.0]; RU, resonance units; scFv, single-chain Fvantibody fragment; TPBS, 0.05% (vol/vol) Tween 20 in PBS; TMPBS, 0.05%(vol/vol) Tween 20 in MPBS; TU, transducing units; V_(H), immunoglobulinheavy-chain variable region; V_(K), immunoglobulin kappa light-chainvariable region; V_(L) immunoglobulin light-chain variable region; wt,wild type; CDC: Centers for Disease Control, CMV: cytomegalovirus; Fab:antigen binding fragment of immunoglobulin with variable domain andfirst constant domain, Fc: fragment crystalizable, Fab′₂; fragment,antigen binding; mAb; monoclonal antibody, FACS: fluorescence activatedcell sorting, LD₅₀; lethal dose 50%; MFI: mean fluorescent intensity,MLA: mouse lethality assay; PCR: polymerase chain reaction, SD-CAA:selective growth dextrose casamino acids media, SG-CAA media: selectivegrowth galactose casamino acids media; AgaII; yeast agglutinin receptor,pM; picomolar, fM; femtomolar, IU; International Unit, CHO; Chinesehamster ovary cells.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are usually in the natural “L” isomeric form. However, residuesin the “D” isomeric form can be substituted for any L-amino acidresidue, as long as the desired functional property is retained by thepolypeptide. In addition, the amino acids, in addition to the 20“standard” amino acids, include modified and unusual amino acids, whichinclude, but are not limited to those listed in 37 CFR (§1.822(b)(4)).Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates either a peptide bond to afurther sequence of one or more amino acid residues or a covalent bondto a carboxyl or hydroxyl end group. However, the absence of a dashshould not be taken to mean that such peptide bonds or covalent bond toa carboxyl or hydroxyl end group is not present, as it is conventionalin representation of amino acid sequences to omit such.

The term “antibody” (also used interchangeably with “immunoglobulin”)encompasses polyclonal and monoclonal antibody preparations where theantibody may be of any class of interest (e.g., IgM, IgG, and subclassesthereof), as well as preparations including hybrid antibodies, alteredantibodies, F(ab′)₂ fragments, F(ab) molecules, Fv fragments, singlechain fragment variable displayed on phage (scFv), single chainantibodies, single domain antibodies, chimeric antibodies, humanizedantibodies, and functional fragments thereof which exhibit immunologicalbinding properties of the parent antibody molecule. The antibodies maybe conjugated to other moieties, and/or may be bound to a support (e.g.,a solid support), such as a polystyrene plate or bead, test strip, andthe like.

Immunoglobulin polypeptides include the kappa and lambda light chainsand the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta, epsilon and muheavy chains or equivalents in other species. Full-length immunoglobulin“light chains” (usually of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 150 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

An immunoglobulin light or heavy chain variable region is composed of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been defined (see, “Sequences ofProteins of Immunological Interest,” E. Kabat et al., U.S. Department ofHealth and Human Services, (1991 and Lefranc et al. IMGT, theinternational ImMunoGeneTics information System®. Nucl. Acids Res.,2005, 33, D593-D597)). A detailed discussion of the IMGTS system,including how the IMGTS system was formulated and how it compares toother systems, is provided on the World Wide Web atimgt.cines.fr/textes/IMGTScientificChart/Numbering/IMGTnumberingsTable.html.The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs. The CDRs are primarily responsible for binding to an epitope of anantigen. All CDRs and framework provided by the present disclosure aredefined according to Kabat et al, supra, unless otherwise indicated.

An “antibody” thus encompasses a protein having one or more polypeptidesthat can be genetically encodable, e.g., by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies encompass intact immunoglobulins as well as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to VH-CHI by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies, including, but are not limited to, Fab′₂,IgG, IgM, IgA, scFv, dAb, nanobodies, unibodies, and diabodies.

Antibodies and fragments of the present disclosure encompass those thatare bispecific. Bispecific antibodies or fragments can be of severalconfigurations. For example, bispecific antibodies may resemble singleantibodies (or antibody fragments) but have two different antigenbinding sites (variable regions). Bispecific antibodies may be producedby chemical techniques (Kranz et al. (1981) Proc. Natl. Acad. Sci., USA,78: 5807), by “polydoma” techniques (see, e.g., U.S. Pat. No.4,474,893), or by recombinant DNA techniques. Bispecific antibodies mayhave binding specificities for at least two different epitopes, at leastone of which is an epitope of BoNT. The BoNT binding antibodies andfragments can also be heteroantibodies. Heteroantibodies are two or moreantibodies, or antibody binding fragments (e.g., Fab) linked together,each antibody or fragment having a different specificity.

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

A 1B10 antibody refers to an antibody expressed by clone 1B10 or to anantibody synthesized in other manners, but having the same CDRs andoptionally, the same framework regions as the antibody expressed byclone 1B10. Similarly, antibodies 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23,2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2,B11.A5, B11.E9, B11.F7, B6.1, B11E8, 4E17.1, 4E16.1, 3E6.1, B12.1,B11.H12 B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4,ING1.1C1, ING1.5B1, ING1.2B10, ING1.3C2, and the like refer toantibodies expressed by the corresponding clone(s) and/or to antibodiessynthesized in other manners, but having the same CDRs and optionally,the same framework regions as the referenced antibodies.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(D)) of the interaction, wherein a smallerK_(D) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (k_(on)) andthe “off rate constant” (k_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.The ratio of k_(off)/k_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the equilibrium dissociationconstant K_(D) (see, generally, Davies el al. Ann. Rev. Biochem.1990,59: 439-15 473).

A “BoNT-neutralizing antibody” refers to an antibody that binds to oneor more Botulinum neurotoxin(s) (e.g., BoNT/B1, BoNT/B2, etc.) and thatby so-binding reduces the toxicity and/or circulating level of that BoNTneurotoxin. Thus, for example the term “BoNT/B-neutralizing antibody”,as used herein refers to an antibody that specifically binds to a BoNT/Bpolypeptide (e.g, a BoNT/B1 polypeptide). An exemplary antibody may bindto an H_(C) domain of a BoNT/B polypeptide and reduces the toxicityand/or circulating level of the BoNT/B polypeptide. Reduced toxicity canbe measured as an increase in the time that paralysis developed and/oras a lethal dosage (e.g., LD₅₀) as described herein. Antibodies derivedfrom BoNT-neutralizing antibodies include, but are not limited to, theantibodies whose sequence is expressly provided herein.

Antibodies derived from BoNT-neutralizing antibodies have a bindingaffinity of about 1.6×10⁻⁸ or better and can be derived by screeninglibraries of single chain Fv fragments displayed on phage or yeastconstructed from heavy (V_(H)) and light (V_(L)) chain variable regiongenes obtained from mammals, including mice and humans, immunized withbotulinum toxoid, toxin, or BoNT fragments. Antibodies can also bederived by screening phage or yeast display libraries in which a knownBoNT-neutralizing variable heavy (V_(H)) chain is expressed incombination with a multiplicity of variable light (V_(L)) chains orconversely a known BoNT-neutralizing variable light chain is expressedin combination with a multiplicity of variable heavy (V_(H)) chains.BoNT-neutralizing antibodies also include those antibodies produced bythe introduction of mutations into the variable heavy or variable lightcomplementarity determining regions (CDR1, CDR2 or CDR3) as describedherein. Finally BoNT-neutralizing antibodies include those antibodiesproduced by any combination of these modification methods as applied tothe BoNT-neutralizing antibodies described herein and their derivatives.

An “epitope” is a site on an antigen (e.g. BoNT) to which an antibodybinds. Epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a spatial conformation. Methods of determiningspatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

A neutralizing epitope refers to the epitope specifically bound by aneutralizing antibody.

“Isolated” refers to an entity of interest that is in an environmentdifferent from that in which the compound may naturally occur. An“isolated” compound is separated from all or some of the components thataccompany it in nature and may be substantially enriched. “Isolated”also refers to the state of a compound separated from all or some of thecomponents that accompany it during manufacture (e.g., chemicalsynthesis, recombinant expression, culture medium, and the like).

A single chain Fv (“scFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which may be expressed from a nucleic acidincluding V_(H)- and V_(L)-encoding sequences either joined directly orjoined by a peptide-encoding linker (Huston, et al. (1988) Proc. Nat.Acad. Sci. USA, 85: 5879-5883). A number of structures are available forconverting the light and heavy polypeptide chains from an antibody Vregion into an scFv molecule which will fold into a three dimensionalstructure substantially similar to the structure of an antigen-bindingsite. See, e.g. U.S. Pat. Nos. 5,091,513 and 5,132,405 and 4,956,778.

Recombinant design methods may be used to develop suitable chemicalstructures (linkers) for converting two heavy and light polypeptidechains from an antibody variable region into a scFv molecule which willfold into a three-dimensional structure that is substantially similar tonative antibody structure.

Design criteria include determination of the appropriate length to spanthe distance between the C-terminal of one chain and the N-terminal ofthe other, wherein the linker is generally formed from small hydrophilicamino acid residues that do not tend to coil or form secondarystructures. Such methods have been described in the art. See, e.g., U.S.Pat. Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No.4,946,778 to Ladner et al.

In this regard, the first general step of linker design involvesidentification of plausible sites to be linked. Appropriate linkagesites on each of the V_(H) and V_(L) polypeptide domains include thosewhich will result in the minimum loss of residues from the polypeptidedomains, and which will necessitate a linker comprising a minimum numberof residues consistent with the need for molecule stability. A pair ofsites defines a “gap” to be linked. Linkers connecting the C-terminus ofone domain to the N-terminus of the next generally comprise hydrophilicamino acids which assume an unstructured configuration in physiologicalsolutions and may be free of residues having large side groups whichmight interfere with proper folding of the V_(H) and V_(L) chains. Thus,suitable linkers generally comprise polypeptide chains of alternatingsets of glycine and serine residues, and may include glutamic acid andlysine residues inserted to enhance solubility. One particular linkerhas the amino acid sequence (Gly₄Ser)₃ (SEQ ID NO:109). Anotherparticularly preferred linker has the amino acid sequence comprising 2or 3 repeats of [(Ser)₄Gly] (SEQ ID NO:110), such as [(Ser)₄Gly]₃ (SEQID NO:111), and the like. Nucleotide sequences encoding such linkermoieties can be readily provided using various oligonucleotide synthesistechniques known in the art (see, e.g., Sambrook, supra.).

The phrase “specifically binds to” or “specifically immunoreactivewith”, when referring to an antibody refers to a binding reaction whichis determinative of the presence of the protein in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein and do not bind in a significant amount to otherproteins present in the sample. Specific binding to a protein under suchconditions may require an antibody that is selected for its specificityfor a particular protein. For example, BoNT/B-neutralizing antibodiescan be raised to BoNT/B protein(s) that specifically bind to BoNT/Bprotein(s), and not to other proteins present in a tissue sample. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substituting oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

DETAILED DESCRIPTION

This disclosure provides antibodies that specifically bind to botulinumneurotoxin, with those that bind to botulinum neurotoxin serotype B andE being of particular interest, as well as antibodies that bind otherbotulinum neurotoxin serotypes (e.g., A). Botulinum neurotoxin isproduced by the anaerobic bacterium Clostridium botulinum. Botulinumneurotoxin poisoning (botulism) arises in a number of contextsincluding, but not limited to food poisoning (food borne botulism),infected wounds (wound botulism), “infant botulism” from ingestion ofspores and production of toxin in the intestine of infants, and as achemical/biological warfare agent. Botulism is a paralytic disease thattypically begins with cranial nerve involvement and progresses caudallyto involve the extremities. In acute cases, botulism can prove fatal.

There are multiple subtypes of various BoNT serotypes. There are alsomany antibodies that bind, for example the BoNT/A1 subtype but will notbind the BoNT/A2 subtype, and so forth.

The present disclosure is related to the discovery that particularlyefficient neutralization of a botulism neurotoxin (BoNT) subtype isachieved by the use of neutralizing antibodies that bind two, three, ormore subtypes of the particular BoNT serotype with high affinity. Thismay be accomplished by using two, three, four, or more differentantibodies directed against each of the subtypes, or alternatively, bythe use of antibodies that are cross-reactive for different BoNTsubtypes, or by bispecific or polyspecific antibodies with specificitiesfor two, three, or four or more BoNT epitopes, and/or serotypes, and/orsubtypes.

It was discovered that combining neutralizing antibodies increases thepotency of the antibody composition dramatically. This increase makes itpossible to generate a multi-antibody, and/or multi-specific antibodiesof the required potency for therapeutic use. The high potency of theantibody combinations can allow vialling of an extremely low dose oftotal antibody as a therapeutic dose, resulting in lower manufacturingcosts. As one combines two, three or four monoclonal antibodies, theparticular BoNT epitope that is recognized becomes less important. Thus,compositions containing at least two, or at least three high affinityantibodies that bind overlapping or non-overlapping epitopes on the BoNTare contemplated herein.

Thus, compositions contemplated herein may include two or more, three ormore, four or more, five or more different antibodies selected from theantibodies described herein (see, e.g., FIGS. 1, 2, and 11) and/orantibodies comprising one or more CDRs from these antibodies, and/or oneor more antibodies comprising mutants or derivatives of theseantibodies. The composition may include antibodies selected from thegroup consisting of 1B10.1, 2B18.1, B11.E8, and optionally B6.1 andB8.1. The compositions may also contain antibodies 4E17.1 together withtwo of the following: 3E2, 3E6.1, 3E6.2, 4E16.1.

Compositions contemplated herein may include monovalent BoNT/Bantitoxins (e.g. comprising B12.1, B11.E8, B6.1, 2B18.1 and 1B10.1).Compositions may also include monovalent BoNT/E antitoxins (e.g.comprising 3E2, 3E6.1, 3E6.2, 4E16.1, and 4E17.1). Compositionscontaining trivalent BoNT/A, BoNT/B and BoNT/E toxins (e.g. comprisingantibodies selected from those described in PCT Pub. Nos. WO 07/094754,WO 05/016232, and 09/008916, and B12.1, B11.E8, B6.1, 2B18.1, 1B10.1,3E2, 3E6.1, 3E6.2, 4E16.1, and 4E17.1) are also contemplated.

As indicated above, the antibodies provided by the present disclosurebind to one or more botulinum neurotoxin type B, E, and in certaininstances Bont/A subtypes, and, in some embodiments, can neutralize theneurotoxin. Neutralization, in this context, refers to a measurabledecrease in the toxicity and/for circulating level of the targetneurotoxin. Such a decrease in toxicity can be measured in vitro by anumber of methods well known to those of skill in the art. One suchassay involves measuring the time to a given percentage (e.g., 50%)twitch tension reduction in a hemidiaphragm preparation. Toxicity can bedetermined in vivo, e.g. as an LD₅₀ in a test animal (e.g. mouse)botulinum neurotoxin type A in the presence of one or more putativeneutralizing antibodies. The neutralizing antibody or antibodycombination can be combined with the botulinum neurotoxin prior toadministration, or the animal can be administered the antibody prior to,simultaneous with, or after administration of the neurotoxin. The rateof clearance of BoNT mediated by a test antibody, or combination of testantibodies, can be measured (e.g. in mice) by administering labeled BoNT(e.g. radiolabeled BoNT/A) and measuring the levels of BoNT in the serumand the liver over time in the presence or absence of test antibody orantibodies (see, e.g., Ravichandran et al. (2006) J Pharmacol Exp Ther318: 1343-1351 (2006).

As the antibodies of the present disclosure act to neutralize botulinumneurotoxins, they are useful in the treatment of pathologies associatedwith botulinum neurotoxin poisoning. The treatments essentially compriseadministering to the poisoned organism (e.g. human or non-human mammal)a quantity of one or more neutralizing antibodies sufficient toneutralize (e.g. mitigate or eliminate) symptoms of BoNT poisoning.

Such treatments are most desired and efficacious in acute cases (e.g.where vital capacity is less than 30-40 percent of predicted and/orparalysis is progressing rapidly and/or hypoxemia with absolute orrelative hypercarbia is present. These antibodies can also be used totreat early cases with symptoms milder than indicated (to preventprogression) or even prophylactically (a use the military envisions forsoldiers going in harm's way). Treatment with the neutralizing antibodycan be provided as an adjunct to other therapies (e.g. antibiotictreatment).

The antibodies provided by this disclosure can also be used for therapid detection/diagnosis of botulism (type B, E, or A toxin(s)) andthereby supplement and/or replace previous laboratory diagnostics.

This disclosure also provides the epitopes specifically bound bybotulinum neurotoxin antibodies described herein. These epitopes can beused to isolate, and/or identify and/or screen for other antibodies BoNTneutralizing antibodies as described herein.

I. Potency of Botulinum Neurotoxin (BoNT)-Neutralizing Antibodies.

Without being bound to a particular theory, it is believed that thecurrent antitoxins used to treat botulism (horse and human) have apotency of about 5000 mouse LD₅₀s/mg (human) and 55,000 mouse LD₅₀s/mg(horse).

Based on calculation, a commercially desirable antitoxin may generallyhave a potency greater than about 10,000 to 100,000 LD₅₀s/mg.Combinations of the antibodies described herein (e.g., two or threeantibodies) can meet this potency. Thus, this disclosure providesantibodies and/or antibody combinations that neutralize at least about10,000 mouse LD₅₀s/mg of antibody, preferably at least about 15,000mouse LD₅₀s/mg of antibody, more preferably at least about 20,000 mouseLD₅₀s/mg of antibody, and most preferably at least about 25,000 mouseLD₅₀s/mg of antibody.

II. Botulinum Neurotoxin (BoNT)-Neutralizing Antibodies.

BoNT neutralizing antibodies may be selected based on their affinity toone or more BoNT subtypes. A number of subtypes are known for each BoNTserotype. Thus, for example, BoNT/A subtypes include, but are notlimited to, BoNT/A1, BoNT/A2, BoNT/A3, and the like (see, e.g., FIG. 3).It is also noted, for example, that the BoNT/A1 subtype includes, but isnot limited to 62A, NCTC 2916, ATCC 3502, and Hall hyper (Hall Allergan)and are identical (99.9-100% identity at the amino acid level.) and havebeen classified as subtype A1. The BoNT/A2 sequences (Kyoto-F andFRI-A2H) (Willems, et al. (1993) Res. Microbiol. 144:547-556) are 100%identical at the amino acid level. Another BoNT/A subtype, (that we arecalling A3) is produced by a strain called Loch Maree that killed anumber of people in an outbreak in Scotland.

Similarly, as shown in FIG. 3, a number of subtypes are also known forserotypes B, C, E, and F. Using, the methods described herein, it wasdiscovered that high affinity antibodies that are cross-reactive withtwo or more subtypes within a serotype can also be produced (e.g.,selected/engineered). Moreover, without being bound to a particulartheory, it appears that these cross-reactive antibodies can be moreefficient in neutralizing Botulinum neurotoxin, particularly when usedin combination one or more different neutralizing antibodies.

The sequences of the variable heavy (V_(H)) and variable light (V_(L))domains for a number of prototypical BoNTB and BoNT/E antibodies areillustrated in Tables 1-5, and in FIGS. 1-2 and 11. The relationship ofcertain antibodies specific for BoNT/E and BoNT/B disclosed herein withrespect to the clonal lineage of the first lead selected is provided inFIG. 10.

The antibodies of the present disclosure can be used individually,and/or in combination with each other, and/or in combination with otherknown anti-BoNT antibodies (see, e.g., Application Pub. No: 20080124328,filed on Jan. 26, 2006, application Ser. No. 09/144,886, filed on Aug.31, 1998, Applicaiont Pub. No. 20040175385, filed on Aug. 1, 2003,60/942,173, filed on Jun. 5, 2007 and PCT Pub. Nos. WO 07/094754, WO05/016232, and WO 09/008916, which are incorporated herein by referencefor all purposes). These antibodies can be used individually, and/or incombination with each other, and/or in combination with other knownanti-BoNT antibodies to form bispecific or polyspecific antibodies.

TABLE 1Deduced protein sequences of heavy chain variable regions (VH) ofBoNT/E binders. Sequence identification numbers next to each clone name identifies an aminoacid sequences for the full length heavy chain variable region. VHClone/ Gene Framework Framework  Framework Framework Family 1 CDR1 2CDR2 3 CDR3 4 2A10 QVQLQQS RYTIT WVRQAPG GIIPIFDKA  RVTFTAD YSRGY WGPGTLVH1 GAEVKKP (SEQ ID QGLEWM NYAQKFQ ASTSTAY VHFDY VTVSS GSSVKVS NO: 113)G S MELGSLR (SEQ ID  (SEQ ID CKASGGT (SEQ ID (SEQ ID PEDTAVY NO: 117)NO: 118) FT NO: 114) NO: 115) YCAA (SEQ ID (SEQ ID NO: 112) NO: 116) 3E1QVQLVES NSGFT WVRQVPG GIIPMFGP RVTITADE DQGEY WGEGTT VH1 GAEVKKP(SEQ ID  QGLEWM ANYAQKF STRMVYM TVGML VTVSS GSSVKVS NO: 120) G QGELRSLRSE LYYAM (SEQ ID CKASGGT (SEQ ID (SEQ ID DTAVYYC DV NO: 125) FSNO: 121) NO: 122) AR (SEQ ID (SEQ ID (SEQ ID NO: 124) NO: 119) NO: 123)3E2 QVQLQES KYAIT WLRQAPG GITPIFATT RVMITAD SPRGGI WGQGTM VH1 GAEVKKP(SEQ ID QGFLWMG NYAQKFQ EVTSTVY VGTFDT VTVSS GSSVKVS NO: 127) (SEQ ID GMDLSSLG (SEQ ID (SEQ ID CKASGGD NO: 128) (SEQ ID SEDTAIYF NO: 131)NO: 132) LN NO: 129) CAK (SEQ ID (SEQ ID NO: 126) NO: 130) 3E3 QVQLVESNYNMN WVRQAPG SISDGGSY RFTISRDN DEMVH WGQGTT VH3 GGGLVKP (SEQ ID KGLEWVSRYYAYSV TKNSLYL GILVYY VTVSS GESLRLSC NO: 134) (SEQ ID KG QMNSLRA GMDV(SEQ ID AASGFTFS NO: 135) (SEQ ID EDTALYY (SEQ ID NO: 139) (SEQ IDNO: 136) CAR NO: 138) NO: 133) (SEQ ID NO: 137) 3E4 QVQLQES SDAMSWVRQAPG AILPSGEA RFTISRHS DSYHS WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVATYYADSV SKNTLYL RLAAF VTVSS GGSLRLSC NO: 141) (SEQ ID KG QMNSLRA DI(SEQ ID GASGFTFS NO: 142) (SEQ ID DDTAVYY (SEQ ID NO: 146) (SEQ IDNO: 143) CAR NO: 145) NO: 140) (SEQ ID NO: 144) 3E4.1 QVQLQES SDAMSWVRQAPG AILPSGEA RFTISRHS DSYHS WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVATYYADSV SKNTLYL RLAAF VTVSS GGSLRLSC NO: 148)  (SEQ ID KG QMNSLRA DI(SEQ ID GASGFTFS NO: 149) (SEQ ID DDTAVYY (SEQ ID NO: 153) (SEQ IDNO: 150) CAR NO: 152) NO: 147) (SEQ ID NO: 151) 3E5 QVQLVQS DFYMSWIRQAPG YIGSSGSA RFTISRDN VASRY WGQGTM VH3 GGGVVQP (SEQ ID KGLEWVSLQYADSV DKNVLYL HDVLT VTVSS GRPLRLSC NO: 155)  (SEQ ID KG QMTSLRA DGFDI(SEQ ID AASTFNFR NO: 156) (SEQ ID EDTAVYY (SEQ ID NO: 160) (SEQ IDNO: 157) CAR NO: 159) NO: 154) (SEQ ID NO: 158) 3E6 QVQLVQS SYAMHWVRQAPG VISYDGN RFTISRDN ARLCTS WGQGTL VH3 GGGVVQP (SEQ ID KGLEWVAKKYYADS SKNTLYL TSCYW VTVSS GKSLRLSC NO: 162) (SEQ ID VKG QMNSLRA TFDP(SEQ ID AASGFTFS NO: 163) (SEQ ID EDAAVFY (SEQ ID NO: 167) (SEQ IDNO: 164) CAR NO: 166) NO: 161) (SEQ ID NO: 165) 3E6.1 QVQLVQS SYAMHWVRQAPG VISYDGN RFTISRDN ARLCTS WGQGTL VH3 GGGVVQP (SEQ ID KGLEWVAKKYYADS SKNTLYL TSCYW VTVSS GKSLRLSC NO: 169) (SEQ ID VKG QMNSLRA TFDP(SEQ ID AASGFTFS NO: 170) (SEQ ID EDAAVFY (SEQ ID NO: 174) (SEQ IDNO: 171) CAR NO: 173) NO: 168) (SEQ ID NO: 172) 4E11 QVQLVQS GYSFNWVRQAPG YMSSGGSI RFTISRDN GPPGRP WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVAKNYADSV AKNSLYL NDAFDI VTVSS GGSLRLSC NO: 176) (SEQ ID KG QVNSLRD(SEQ ID (SEQ ID AASGFRFS NO: 177) (SEQ ID EDTALYY NO: 180) NO: 181)(SEQ ID NO: 178) CAR NO: 175) (SEQ ID NO: 179) 4E13 EVQLVQS SYAMTWVRQAPG SISVSGDS RFTISRDN GLSKA WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVSTYYADSV SKNTVSL DLFGM VTVSS GGSLRLSC NO: 183) (SEQ ID KG QMNSLRA DV(SEQ ID AASGFTFS NO: 184) (SEQ ID EDTALYY (SEQ ID NO: 188) (SEQ IDNO: 185) CAK NO: 187) NO: 182) (SEQ ID NO: 186) 4E16 QVQLQES DYYWSWIRQPPG YIYYSGST RVTISVDT HTSGW WGQGTM VH4 GPGLVKPS (SEQ ID KGLEWIGNYNPSLKS SKNQFSLN SGGAF VTVSS ETLSLTCS NO: 190)  (SEQ ID (SEQ ID LSSVTAADI (SEQ ID VSGVSIS NO: 191) NO: 192) DTAVYYC (SEQ ID NO: 195) (SEQ ID ARNO: 194) NO: 189) (SEQ ID NO: 193) 4E16.1 QVQLQES DYYWS WIRQPPG YIYYSGSTRVTISVDT HTSGW WGQGTM VH4 GPGLVKPS (SEQ ID KGLEWIG NYNPSLKS SKNQFSLNSGGAF VTVSS ETLSLTCS NO: 197)  (SEQ ID (SEQ ID LSSVTAA DI (SEQ IDVSGVSIS NO: 198) NO: 199) DTAVYYC (SEQ ID NO: 202) (SEQ ID AR NO: 201)NO: 196) (SEQ ID NO: 200) 4E17 EVQLVQS SHWMT WVRQAPG NINLDGTE RFTVSRDLQWGG WGQGTL VH3 GGNLVQP (SEQ ID QGLEWVA KFYVDSV NRKSSVFL YNGWL VTVSSGGSLRLSC NO: 204)  (SEQ ID KG QMNNLRV SP (SEQ ID AATGPIG NO: 205)(SEQ ID DDTAVYY (SEQ ID NO: 209) (SEQ ID NO: 206) CAR NO: 208) NO: 203)(SEQ ID NO: 207) 4E17.1  EVQLVRS SHWMT WVRQAPG NINLDGTE RFTVSRD LQWGGWGQGTL VH3 GGNLVQP (SEQ ID QGLEWVA KFYVDSV NRKSSVFL YNGWL VTVSS GGSLRLSCNO: 211) (SEQ ID KG QMNNLRV SP (SEQ ID AATGPIG NO: 212) (SEQ ID DDTAVYY(SEQ ID NO: 216) (SEQ ID NO: 213) CAR NO: 215) NO: 210) (SEQ ID NO: 214)3E6.2 QVQLVQS GYAMH WVRQAPG VISYDGN RFTISRDN ARLCTS WGQGTL VH3 GGGVVQP(SEQ ID KGLEWVA KKYYADS SKNTLYL TSCYW VTVSS GKSLRLSC NO: 218)  (SEQ IDVKG (SEQ QMNSLRA TFDP (SEQ ID AASGFAF NO: 219) ID NO: 220) EDAAVFY(SEQ ID NO: 223) G (SEQ ID CAR (SEQ NO: 222) NO: 217) ID NO: 221)4E17.4  EVQLVRS QHWMT WVRQAPG NINLDGTE RFTVSRD LQWGG WGQGTL VH3 GGNLVQP(SEQ ID QGLEWVA KFYVDSV NRKSSVFL YNGWL VTVSS GGSLRLSC NO: 225) (SEQ IDKG (SEQ QMNNLRV SP (SEQ (SEQ ID AATGPIT NO: 226) ID NO: 227) DDTAVYY IDNO: 230) (SEQ ID CAR (SEQ NO: 229) NO: 224) ID NO: 228) 4E17.6  EVQLVRSQHWMT WVRQAPG NINLDGTE RFTVSRD LQWGG WGQGTL VH3 GGNLVQP (SEQ ID QGLEWVAKFYVDSV NRKSSVFL YNGWL VTVSS GGSLRLSC NO: 232) (SEQ ID KG (SEQ QMNNLRVSP (SEQ (SEQ ID AATGPIT NO: 233) ID NO: 234) DDTAVYY ID NO: 237) (SEQ IDCAR (SEQ NO: 236) NO: 231) ID NO: 235)

TABLE 2 Deduced protein sequences of light chain variable regions (VL) of BoNT/E binders.Sequence identification numbers next to each clone name identifies an aminoacid sequences for the full length light chain variable region.VL/Clone/ Gene Framework Framework Framework Framework Family 1 CDR1 2CDR2 3 CDR3 4 2A10 DIVMTQSP WASQG WYQQKPG AASTLQ GVPSRFSGS QQLNSY FGGGTKVK1 SFLSASVG ISSYLA KAPKLLIY S (SEQ GSGTEFTLTI PLT (SEQ VDIKR DRVTITC(SEQ ID (SEQ ID ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 239) NO: 240)NO: 241) TYYC (SEQ NO: 243) NO: 244) NO: 238) ID NO: 242) 3E1 EIVLTQSPRASQGI WYQHKA AASSLQ GVPSRFSGS QQYNSY FGGGTK VK1 DSLSASVG SGYLA GKAPKLLIS (SEQ GYGTEFTLTI PFT (SEQ VEIKR DRVTITC (SEQ ID Y (SEQ ID ID SSLQPDDFAID (SEQ ID (SEQ ID NO: 246) NO: 247) NO: 248) TYYC (SEQ NO: 250)NO: 251) NO: 245) ID NO: 249) 3E2 EIVLTQSP RTSQSI WYQQKA AASTLHGVPSRFSGS QQSYSIP FGGGTK VK1 SFLSAFVG NNYLN GKAPKLLI T (SEQ GSGTEFTLTILT (SEQ VEIKR DRVTITC (SEQ ID Y (SEQ ID ID SSLQPEDFA ID (SEQ ID (SEQ IDNO: 253) NO: 254) NO: 255) TYYC (SEQ NO: 257) NO: 258) NO: 252)ID NO: 256) 3E3 DIVMTQSP RASQSF WYQQKPG AASSRA GVPTGSVAD QQSYST FGGGTKVK3 DSLSASVG SSSYLA QAPRLLIY A (SEQ GSGTDFTLTI PYT VEIKR DSVTITC (SEQ ID(SEQ ID ID SGLQPEDFA (SEQ ID (SEQ ID (SEQ ID NO: 260) NO: 261) NO: 262)AYYC (SEQ NO: 264) NO: 265) NO: 259) ID NO: 263) 3E4 DIVMTQSP RASQSIWYQQKPG KASSLE GVPSRFSGS QQYNA FGGGTK VK1 SFLSAFVG SNWLA KAPKVLIY N (SEQGSGTDFTLTI YPLT VEIKR DRVTITC (SEQ ID (SEQ ID ID TSLQPDDFA (SEQ ID(SEQ ID (SEQ ID NO: 267) NO: 268) NO: 269) TYYC (SEQ NO: 271) NO: 272)NO: 266) ID NO: 270) 3E4.1 EIVLTQSP RASQRI WYQQKPG KAFSLE GVPSRFSGSQQYDSY FGQGTKL VK1 STLSASVG GSWLA KAPNPLIY S (SEQ RSGTEFTLTI PYT EIKRDRVAITC (SEQ ID (SEQ ID ID SSLQPDDFA (SEQ ID (SEQ ID (SEQ ID NO: 274)NO: 275) NO: 276) TYFC (SEQ NO: 278) NO: 279) NO: 273) ID NO: 277) 3E5DVVMTQS QASQDI WYQQKPG DASNLE GVPSRFSGS QQYDPL FGGGTK VK1 PSSLSASIGSNRLN KVPKLLIS T (SEQ GSGTDFTFTI LT (SEQ VEIKR DRVTFTC (SEQ ID (SEQ IDID SSLQPEDIAT ID (SEQ ID (SEQ ID NO: 281) NO: 282) NO: 283) YYC (SEQ IDNO: 285) NO: 286) NO: 280) NO: 284) 3E6 DIQMTQSP RASQGI WYQQKSG AASSLQGVPSRFSGS QQAYRT FGGGTK VK1 SSVSASVG SSWLA QAPTLLIY S (SEQ GSGTDFTLIIPIT (SEQ VEIKR DTVTISC (SEQ ID (SEQ ID ID SSLQPEDFA ID (SEQ ID (SEQ IDNO: 288) NO: 289) NO: 290) TYYC (SEQ NO: 292) NO: 293) NO: 287)ID NO: 291) 3E6.1 DIQMTQSP QASQDI WYQQKPG AASSLQ GVPSRFSGS QQSYNTFGQGTKL VK1 SSVSASVG SNYLN KAPKLLIY S (SEQ GSGTDFTLTI PPT (SEQ EIKRDRVSITC (SEQ ID (SEQ ID ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 295)NO: 296) NO: 297) TYYC (SEQ NO: 299) NO: 300) NO: 294) ID NO: 298) 4E11ASVLTQD QGDSL WYQQKPG GKSNRP GIPDRFSGSS NSRDST FGGGTK VL3 PAVSVAL RSYYAQAPVLVIY S (SEQ SGNTASLTIT GNQL VTVLG GQTVRITC S (SEQ ID (SEQ ID IDGAQAEDEA (SEQ ID (SEQ ID (SEQ ID NO: 302) NO: 303) NO: 304) DYYC (SEQNO: 306) NO: 307) NO: 301) ID NO: 305) 4E13 AELTQDP QGDSL WYQQKPG GENSRPGIPDRFSGSS NSPDSS FGGGTK VL3 AVSVALG RSYYA QAPVLVIY S (SEQ SGNTASLTIGIHLV VTVLG QTVRITC S (SEQ ID (SEQ ID ID AGAQAEDE (SEQ ID (SEQ ID(SEQ ID NO: 309) NO: 310) NO: 311) ADYYC (SEQ NO: 313) NO: 314) NO: 308)ID NO: 312) 4E16 EIVLTQSP KSSQSV WYQQKPG WASTRE GVPDRFSGS HQYYSS FGQGTKLVK4 DSLAVSL LYSSN QPPKLLFY S (SEQ GSGTDFTLTI PLT (SEQ EIKR GERATINCNKNYL (SEQ ID ID SSLQAEDVA ID (SEQ ID (SEQ ID A (SEQ NO: 317) NO: 318)VYYC (SEQ NO: 320) NO: 321) NO: 315) ID NO: 316) ID NO: 319) 4E16.1EIVLTQSP KSSQSV WYQQKPG WASTRE GVPDRFSGS QQYYSR FGQGTKL VK4 NSLAVSLLYSGN QPPKLLIY S (SEQ GSETDFTLTI WT (SEQ EIKR GERATIRC NKNYI (SEQ ID IDSSLRAEDVA ID (SEQ ID (SEQ ID A (SEQ NO: 324) NO: 325) LYYC (SEQ NO: 327)NO: 328) NO: 322) ID NO: 323) ID NO: 326) 4E17 DIVMTQSP RASQSI WYQQKPGGTSNLQ GVPSGFSGS QETYST FGQGTKL VK1 SSVSASVG SSYLN KAPKLLIY S (SEQGSGTDFTLTI PPT (SEQ EIKR DRVTITC (SEQ ID (SEQ ID ID SSLQPEDFA ID (SEQ ID(SEQ ID NO: 330) NO: 331) NO: 332) TYYC (SEQ NO: 334) NO: 335) NO: 329)ID NO: 333) 4E17.1 DIVMTQSP RASQSI WYQQKPG KASSLA GAPSRFSGS QQSYSIPFGGGTK VK1 SSLSASVG RHYVN KAPKLLIY S (SEQ GSGTDFTLTI LT (SEQ VEIKRDRVTISC (SEQ ID (SEQ ID ID SSLQPDDFA ID (SEQ ID (SEQ ID NO: 337)NO: 338) NO: 339) TYYC (SEQ NO: 341) NO: 342) NO: 336) ID NO: 340) 3E6.2DIQMTQSP QASQDI WYQQKPG AASSLQ GVPSRFSGS QQSYDT FGQGTKL VK1 SSVSASVGSNYLN KAPKLLIY S (SEQ GSGTDFTLTI PPT (SEQ EIKR DRVSITC (SEQ ID (SEQ IDID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 344) NO: 345) NO: 346) TYYC (SEQNO: 348) NO: 349) NO: 343) ID NO: 347) 4E17.4 DIVMTQSP RASQSI WYQQKPGKASSLA GAPSRFSGS QQSYSIP FGGGTK VK1 SSLSASVG RHYVN KAPKLLIY S (SEQGSGTDFTLTI LT (SEQ VEIKR DRVTISC (SEQ ID (SEQ ID ID SSLQPDDFA ID (SEQ ID(SEQ ID NO: 351) NO: 352) NO: 353) TYYC (SEQ NO: 355) NO: 356) NO: 350)ID NO: 354) 4E17.6 DIQMTQSP RASQGI WYQQKPG KASSLE GVPSRFSGS QQLNTYFGQGTK VK1 SFLSASVG SSYLA KAPKLLIY S (SEQ GSGTDFALT PQT VEIKR DRVTITC(SEQ ID (SEQ ID ID ISSLQPEDFA (SEQ ID (SEQ ID (SEQ ID NO: 358) NO: 359)NO: 360) TYYC (SEQ NO: 362) NO: 363) NO: 357) ID NO: 361)

TABLE 3 Deduced protein sequences of heavy chain variable regions (VH) of BoNT/B binders.Sequence identification numbers next to each clone name identifies an aminoacid sequences for the full length heavy chain variable region.VH/Clone/ Gene Framework Framework Framework Framework Family 1 CDR1 2CDR2 3 CDR3 4 A12 EVQLVES SYGMH WVRQAP VIWYD RFTISRDNSK GYSNYD WGQGTTVH3 GGGVVQP (SEQ ID GKGLEW GSNKY NTLYLQMN YYYGM VTVSS GRSLRLSC NO: 365)VA (SEQ ID YADSV SLRAEDTAV DV (SEQ (SEQ ID AASGFTFS NO: 366) KGYYCAR (SEQ ID NO: 370) (SEQ ID (SEQ ID ID NO: 368) NO: 369) NO: 364)NO: 367) 6A12 QVQLVES SYGMH WVRQAP YISSSG RFTISRDNA VSIVGG WGQGTT VH3GGGVVQP (SEQ ID GKGLEW STIYYA KNSLYLQM PYGMD VTVSS GRSLRLSC NO: 372)VS (SEQ DSVKG NSLRAEDTA V (SEQ (SEQ ID AASGFTFS ID (SEQ ID VYYCAR IDNO: 377) (SEQ ID NO: 373) NO: 374) (SEQ ID NO: 376) NO: 371) NO: 375)B1.1 QVQLVQS SYAFT WVRQAP RIVPFL RVTITADKA DKRTYE WGRGTL VH1 GAEVEKP(SEQ ID GQGLEW GVPYY TSTVYMELS YNWNSL VTVSS GSSVKVS NO: 379) MG (SEQTQKFR SLTFDDTAV WF (SEQ (SEQ ID CKASGGS ID G (SEQ YYCAR (SEQ ID NO: 384)FS (SEQ ID NO: 380) ID ID NO: 382) NO: 383) NO: 378) NO: 381) B6 QVQLVQSSFWIA WVRQMP IIYAGD HVNISVDRS HDSRYK WGQGTT VH5 GAEVKKP (SEQ ID GKGLEWSDTRYS TNTAYLQW YFYFGM VTVSS GESLVISC NO: 386) MG (SEQ PSFQG SSLKASDTADV (SEQ (SEQ ID KASGDKD ID (SEQ ID MYYCAR ID NO: 391) TFT (SEQ NO: 387)NO: 388) (SEQ ID NO: 390) ID NO: 385) NO: 389) B6.1 QVQLVQS SFWIA WVRQMPIIYAGD HVNISVDRS HDSRYK WGQGTT VH5 GAEVKKP (SEQ ID GKGLEW SDTRYSTNTAYLQW YFYFGM VTVSS GESLVISC NO: 393) MG (SEQ PSFQG SSLKASDTA DV (SEQ(SEQ ID KASGDKD ID (SEQ ID MYYCAR ID NO: 398) TFT (SEQ NO: 394) NO: 395)(SEQ ID NO: 397) ID NO: 392) NO: 396) B8 QVQLLES SYGMH WVRQAP VIWYDRFTISRDNSK GYSNYD WGQGTT VH3 GGGVVQP (SEQ ID GKGLEW GSNKY DTLYLQMN YYYGMVTVSS GRSLRLSC NO: 400) VA (SEQ YADSV SLRAEDTAV DV (SEQ (SEQ ID AASGFTFSID KG YYCAR (SEQ ID NO: 405) (SEQ ID NO: 401) (SEQ ID ID NO: 403)NO: 404) NO: 399) NO: 402) B8.1 QVQLLES SYGMH WVRQAP VIWYD RFTISRDNSKGYSNYD WGQGTT VH3 GGGVVQP (SEQ ID GKGLEW GSNKY NTLYLQMN YYYGM VTVSSGRSLRLSC NO: 407) VA (SEQ YADSV SLRAEDTAV DV (SEQ (SEQ ID AASGFTFS ID KGYYCAR (SEQ ID NO: 412) (SEQ ID NO: 408) (SEQ ID ID NO: 410) NO: 411)NO: 406) NO: 409) B11 QVQLLQS TYGMH WVRQAP FVSSDG RFTIPRDNA DRYPIDWGQGTT VH3 AGGVVQP (SEQ ID GKGLEW NNKFY KNTLYLQM CSGGSC VTVSS GRSLRLSCNO: 414) VA (SEQ SDSVK NSLETEDTA FSYGMD (SEQ ID AASGFIFR ID G (SEQVYYCAK V (SEQ NO: 419) (SEQ ID NO: 415) ID (SEQ ID ID NO: 413) NO: 416)NO: 417) NO: 418) B11C3 EVQLVES TYGMH WVRQAP FVSSDG RFTIPRDNA DRYPIDWGQGTL VH3 GGGVVQP (SEQ ID GKGLEW NNKFY KNTLYLQM CSGGSC VTVSS GRSLRLSCNO: 421) VA(SEQ SDSVK NSLETEDTA FSYGMD (SEQ ID ATSGFILR ID G (SEQ VYYCAKV(SEQ ID NO: 426) (SEQ ID NO: 422) ID (SEQ ID NO: 425) NO: 420) NO: 423)NO: 424) B11E8 EVQLVQS TYGMH WVRQAP FVSSDG RFTISRDNA DRYPID WGQGTT VH3GGGVVQP (SEQ ID GKGLEW NNKFY KNTLYLQM CSGGSC VTVSS GRSLRLSC NO: 428)VA (SEQ SDSVK NSLETEDTA FSYGMD (SEQ ID AASGFIFR ID G (SEQ MYYCAK V (SEQNO: 433) (SEQ ID NO: 429) ID (SEQ ID ID NO: 427) NO: 430) NO: 431)NO: 432) B12 QVNLRES SYALH WVRQTP LISYDG RFTISRDNSK DRSHYG WGQGTL VH3GGGVVQP (SEQ ID GKGLEW SNKYY NMLYLQMN DYVGYL VTVSS GRSLRLSC NO: 435)VA (SEQ ADSVK SLRAEDTAV DY (SEQ (SEQ ID AASGFTFS ID G (SEQ YYCAK (SEQ IDNO: 440) (SEQ ID NO: 436) ID ID NO: 438) NO: 439) NO: 434) NO: 437)B12.1 QVNLRES SYALH WVRQTP LISYDG RFTISRDNSK DRSHYG WGQGTL VH3 GGGVVQP(SEQ ID GKGLEW SNKYY NMLYLQMN DYVGYL VTVSS GRSLRLSC NO: 442) VA (SEQADSVK SLRAEDTAV DY (SEQ (SEQ ID AASGFTFS ID G (SEQ YYCAK (SEQ IDNO: 447) (SEQ ID NO: 443) ID ID NO: 445) NO: 446) NO: 441) NO: 444)B12.2 QVNLRES SYALH WVRQTP LISYDG RFTISRDNSK DRSHYG WGQGTL VH3 GGGVVQP(SEQ ID GKGLEW SNKYY NMLYLQMN DYVGYL VTVSS GRSLRLSC NO: 449) VA (SEQADSVK SLRAEDTAV DY (SEQ (SEQ ID AASGFTFS ID G (SEQ YYCAK (SEQ IDNO: 454) (SEQ ID NO: 450) ID ID NO: 452) NO: 453) NO: 448) NO: 451) 1B18EVQLVQS AYWM WVRQAP NINLDG RFTVSRDNV LEWGGR WGQGTL VH3 GGGLVQP T (SEQGKGLEW TEIYYL KNSVFLQMS NGWVSP VTVSS GGSRRLSC ID VA (SEQ DSVKG SLRVEDTAV(SEQ ID (SEQ ID AASGFYF NO: 456) ID (SEQ ID YFCAR (SEQ NO: 460) NO: 461)N (SEQ ID NO: 457) NO: 458) ID NO: 459) NO: 455) 2B18.1 QVQLVQS AYWMWVRQAP NINLDG RFTVSRDNV LEWGGR WGQGTL VH3 GGGLVQP T (SEQ GKGLEW TEIYYLKNSVFLQMS NGWVSP VTVSS GGSRRLSC ID VA (SEQ DSVKG SLRVEDTAV (SEQ ID(SEQ ID AASGFYF NO: 463) ID (SEQ ID YFCAR (SEQ NO: 467) NO: 468)N (SEQ ID NO: 464) NO: 465) ID NO: 466) NO: 462) 4B19 QVQLVQS GYYIYWVRQAP WINPNS RVTMTIDTS EWTQL WGQGTT VH1 GAEVKKP (SEQ ID GQGLEW GVTKYTNTAYMEL WSPYDY VTVSS GASVNVS NO: 470) MG (SEQ AQKFQ NRLRADDT (SEQ ID(SEQ ID CKASGYT ID G (SEQ AVYYCAR NO: 474) NO: 475) FT (SEQ ID NO: 471)ID (SEQ ID NO: 469) NO: 472) NO: 473) 1B22 QVQLQES SYSWS WIRQTPG YIYHSGRVTMSVDK TAFYYE WGQGTL VH4 GSRLVKPS (SEQ ID KGLEWIG STYYN SRNQFSLNMNTGPIRC VTVSS QTLSLTCG NO: 477) (SEQ ID PSLKS SSVTAADTA YLDF (SEQ IDVSGGSISS NO: 478) (SEQ ID VYYCAR (SEQ ID NO: 482) S (SEQ ID NO: 479)(SEQ ID NO: 481) NO: 476) NO: 480) 1B10 QVQLVES HYGMH WVRQSP VIWYDRFTISRDND DLTRFH WGPGTT VH3 GGGVVQP (SEQ ID GKGLEW GRNPY KNTLYLQM DTTFGVVTVSS GRSLRLSC NO: 484) VA (SEQ YAASV NSLRAEDTA FEM (SEQ ID AASGFTFS IDKG VYYCVK (SEQ ID NO: 489) (SEQ ID NO: 485) (SEQ ID (SEQ ID NO: 488)NO: 483) NO: 486) NO: 487) 1B10.1 QVQLVES HYGMH WVRQSP VIWYD RFTISRDNDDLTRFH WGPGTT VH3 GGGVVQP (SEQ ID GKGLEW GRNPY KNTLYLQM DTTFGV VTVSS(SEQ ID GRSLRLSC NO: 492) VA (SEQ YAASV NSLRAEDTA FEM (SEQ ID NO: 490)AASGFTFS ID KG VYYCVK (SEQ ID NO: 497) (SEQ ID NO: 493) (SEQ ID (SEQ IDNO: 496) NO: 491) NO: 494) NO: 495) 2B18.2 QVQLVQS AYWM WVRQAP NINLDGRFTVSRDNV LEWGGR WGQGTL VH3 GGGLVQP T (SEQ GKGLEW TEIYYL KNSVFLQMSNGWVSP VTVSS GGSRRLSC ID VA (SEQ DSVKG SLRVEDTAV (SEQ ID (SEQ ID AASGFYFNO: 499) ID (SEQ ID YFCAR (SEQ NO: 503) NO: 504) K (SEQ ID NO: 500)NO: 501) ID NO: 502) NO: 498) 2B18.3 EVQLVES AYWM WVRQAP NINLDGRFTVSRDNV LEWGGR WGQGTL VH3 GGGLVQP T (SEQ GKGLEW TEIYYL KNSVFLQMSNGWLSP VTVSS GGSRRLSC ID VA (SEQ DSVKG SLRVEDTAV (SEQ ID (SEQ ID AASGFYFNO: 506) ID (SEQ ID YFCAR (SEQ NO: 510) NO: 511) N (SEQ ID NO: 507)NO: 508) ID NO: 509) NO: 505) 1B22.4 QVQLQES SYSWS WIRQTPG YIYHSGRVTMSVDK TAFYYE WGQGTL VH4 GSRLVKPS (SEQ ID KGLEWIG STYYN SRNQFSLNMNTGPIRC VTVSS QTLSLTCG NO: 513) (SEQ ID PSLKS SSVTAADTA YLDF (SEQ IDVSGGSISS NO: 514) (SEQ ID VYYCAR (SEQ ID NO: 518) S (SEQ ID NO: 515)(SEQ ID NO: 517) NO: 512) NO: 516) 2B23 EVQLLESG NYPMS WVRQAP SLTASGRFTISRDNSN ALVGRY WGQGTL VH3 GGLVQPG (SEQ ID GKGLAW DNTFY NTLYLQMHDISTGY VTVSS GSLRLSCA NO: 520) VS (SEQ ADSVK SLRAEDTAV YRPVMD (SEQ IDASGFTFS ID G (SEQ YYCAK (SEQ S (SEQ NO: 525) (SEQ ID NO: 521) IDID NO: 523) ID NO: 519) NO: 522) NO: 524) 2B24 QVQLVES VYGMH WVRQAPVISHTG RFSISRDNSN DGPMA WGQGTL VH3 GGGVVQP (SEQ ID GKGLEW SEEYYNTLFLQMNS AIPFYYF VTVSS GRSLRLSC NO: 527) VA (SEQ ADSVK LRPEDTAVYDF (SEQ (SEQ ID AASGLTFS ID G (SEQ YCVK (SEQ ID NO: 532) (SEQ IDNO: 528) ID ID NO: 530) NO: 531) NO: 526) NO: 529) 2B25 QVQLQES SGTFYWIRQHPG YIYYSG RVTLSVDTS GVPIYD WGQGTL VH4 GPGLVKPS WS KDLEWIG TTYYNKNQFSLKVT SSGTYR VTVSS QTLSLSCS (SEQ ID (SEQ ID PSLKS SLTAADTAV GTYFDY(SEQ ID VSGASIT NO: 534) NO: 535) (SEQ ID YHCAR (SEQ (SEQ ID NO: 539)(SEQ ID NO: 536) ID NO: 537) NO: 538) NO: 533) 2B25.1 QVQLQES SGTFYWIRQHPG YIYYSG RVTLSVDTS GVPIYD WGQGTL VH4 GPGLVKPS WS KDLEWIG TTYYNKNQFSLKVT SSGTYR VTVSS QTLSLSCS (SEQ ID (SEQ ID PSLKS SLTAADTAV GTYFDY(SEQ ID VSGASIT NO: 541) NO: 542) (SEQ ID YHCAR (SEQ (SEQ ID NO: 545)(SEQ ID NO: 543) ID NO: 544) NO: 538) NO: 540) 2B26 QVQLVQS NYPMT WVRQAPSVIASG RFTISRDNSK ALVGRY WGQGTT VH3 GGTLVQP (SEQ ID GKGLAW DNTFYNTLYLQMD DISTGY VTVSS GGSLRLSC NO: 547) VS (SEQ ADSVK SLRAEDTAV YRPVLD(SEQ ID AASGFTFS ID G (SEQ YYCAK (SEQ Y (SEQ NO: 552) (SEQ ID NO: 548)ID ID NO: 550) ID NO: 546) NO: 549) NO: 551) 2B27 EVQLQES SNYSW WIRQPPGTMYYS RVTISVDTS RRLLGP WGQGTL VH3 GPGLVKPS A (SEQ KGLEWIG GSTHYKSQLSLKLS SPYYFD VTVSS ETLSVTCA ID (SEQ ID HPSLKS SVTAADTAV Y (SEQ(SEQ ID VSGGSISS NO: 554) NO: 555) (SEQ ID YYCAR (SEQ ID NO: 559)(SEQ ID NO: 556) ID NO: 557) NO: 558) NO: 553) 2B28 EVQLVQS RNAIH WVRQAPLISYDG RFAISRDNA DVSEYG WGQGTL VH3 GGGVVQP (SEQ ID GKGLEW INKYY KNTLFLQMDYVGHF VTVSS GRSLRLSC NO: 561) VA (SEQ ADSVK NSLRAEDTA DY (SEQ (SEQ IDAASGFTFR ID G (SEQ VYYCAR ID NO: 566) (SEQ ID NO: 562) ID (SEQ IDNO: 565) NO: 560) NO: 563) NO: 564) 2B29 QVQLQES SGTYY WIRQHPG YIYYSGRVSMSVDTS GNPQYD WGQGTL VH4 GPGLVKPS WT KGLEWIG TTYYN KNLFSLKMN TSGSYTVTVSS ETLSLTCS (SEQ ID (SEQ ID PSLKS SVTAADTAL GLYFDF (SEQ ID VSGGSINNO: 568) NO: 569) (SEQ ID YYCAR (SEQ (SEQ ID NO: 573) (SEQ ID NO: 570)ID NO: 571) NO: 572) NO: 567) 2B30 EVQLVES RNAIH WVRQAP VISYDG RFAISRDNADVSEYG WGQGTL VH3 GGGVVQP (SEQ ID GKGLEW VNKYY KNTLFLQM DYVGHF VTVSSGRSLRLSC NO: 575) VA (SEQ AASVK NSLRPEDSAI DY (SEQ (SEQ ID AASGFTFR IDG (SEQ YYCAR (SEQ ID NO: 580) (SEQ ID NO: 576) ID ID NO: 578) NO: 579)NO: 574) NO: 577) 4B17.1 EVQLVRS SHWMT WVRQAP NINLDG RFTVSRDNR LQWGGWGQGTL VH3 GGNLVQP (SEQ ID GQGLEW TEKFY KSSVFLQMN YNGWLS VTVSS GGSLRLSCNO: 582) VA (SEQ VDSVK NLRVDDTA P (SEQ (SEQ ID AATGPIG ID G (SEQ VYYCARID NO: 586) (SEQ ID NO: 583) ID (SEQ ID NO: 585) NO: 581) NO: 584)NO: 207) 4B17.1C EVQLVRS SHWMT WVRQAP NINLDG RFTVSRDNR LQWGG WGQGTL VH3GGNLVQP (SEQ ID GQGLEW TEKFY KSSVFLQMN YNGWLS VTVSS GGSLRLSC NO: 588)VA (SEQ VDSVK NLRVDDTA P (SEQ (SEQ ID AATGFPIG ID G (SEQ VYYCAR IDNO: 593) (SEQ ID NO: 589) ID (SEQ ID NO: 592) NO: 587) NO: 590) NO: 591)4B17.1D EVQLVRS SHWMT WVRQAP NINLDG RFTVSRDNR LQWGG WGQGTL VH3 GGNLVQP(SEQ ID GQGLEW TEKFY KSSVFLQMN YNGWLS VTVSS GGSLRLSC NO: 595) VA (SEQVDSVK NLRVDDTA P (SEQ (SEQ ID AATGGPIG ID G (SEQ VYYCAR ID NO: 600)(SEQ ID NO: 596) ID (SEQ ID NO: 599) NO: 594) NO: 597) NO: 598) 4B17.1FEVQLVRS SHWMT WVRQAP NINLDG RFTVSRDNR LQWGG WGQGTL VH3 GGNLVQP (SEQ IDGQGLEW TEKFY KSSVFLQMN YNGWLS VTVSS GGSLRLSC NO: 602) VA (SEQ VDSVKNLRVDDTA P (SEQ (SEQ ID AATGFYIG ID G (SEQ VYYCAR ID NO: 607) (SEQ IDNO: 603) ID (SEQ ID NO: 606) NO: 601) NO: 604) NO: 605) 4B17.1G EVQLVRSSHWMT WVRQAP NINLDG RFTVSRDNR LQWGG WGQGTL VH3 GGNLVQP (SEQ ID GQGLEWTEKFY KSSVFLQMN YNGWLS VTVSS GGSLRLSC NO: 609) VA (SEQ VDSVK NLRVDDTAP (SEQ (SEQ ID AATGFTIG ID G (SEQ ID VYYCAR ID NO: 614) (SEQ ID NO: 610)NO: 611) (SEQ ID NO: 613) NO: 608) NO: 612)

TABLE 4 Deduced protein sequences of light chain variable regions (VL) of BoNT/B binders.Sequence identification numbers next to each clone name identifies an aminoacid sequences for the full length light chain variable region.VL/Clone/   Gene Framework Framework Framework Framework Family 1 CDR1 2CDR2 3 CDR3 4 A12 DIQMTQSP RASQRI WYQQKP AASSL EVPSRFSGS QQSYRP FGGGTKVK1 SSLSASVG SNYLN GKAPKLL QS (SEQ GSGTDFTLTI PLT (SEQ VEIKR DRVTITC(SEQ ID IY (SEQ ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 616) ID NO: 618)TYYC (SEQ NO: 620) NO: 621) NO: 615) NO: 617) ID NO: 619) 6A12 DIQMTQSPRASQGI WYQQKP AASSL GVPSRFSGS QKANSF FGGGTK VK2 SSVSASVG SSWLA GKAPKLLQS (SEQ GSGTDFTLTI PLT (SEQ VEIKR NRVTITC (SEQ ID IY (SEQ ID SSLQPEDFAID (SEQ ID (SEQ ID NO: 623) ID NO: 625) TYYC (SEQ NO: 627) NO: 628)NO: 622) NO: 624) ID NO: 626) B1.1 DVVMTQS RASQSI WYQQKP AASSL GVPSRFSGSQQSYST FGQGTKL VK1 PSSLSASV SSYLN GKAPKLL QS (SEQ GSGTDFTLTI PLT (SEQEIKR GDRVTITC (SEQ ID IY (SEQ ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 630)ID NO: 632) TYYC (SEQ NO: 634) NO: 635) NO: 629) NO: 631) ID NO: 633) B6DVVMTQS QAGQD WYQQKP DASNL GVPSRFSGG QQYDNL FGQGTKL VK1 PSSLSASV ISNFLNGKAPKLL ET (SEQ GSGTHFTFTI PYT EIKR GDRITITC (SEQ ID IR (SEQ IDSSLHPEDIAT (SEQ ID (SEQ ID (SEQ ID NO: 637) ID NO: 639) YFC (SEQ IDNO: 641) NO: 642) NO: 636) NO: 638) NO: 640) B6.1 DIQMTQSP RASQSI WYQQEPSASSLQ GVPSRFSGS QQSYST FGQGTKL VK1 SSLSASVG SSYLN GKAPKLL S (SEQGSGTDFTLTI PPYT EIKR DRVTITC (SEQ ID IY (SEQ ID SSLQPEDFA (SEQ ID(SEQ ID (SEQ ID NO: 644) ID NO: 646) TYYC (SEQ NO: 648) NO: 649)NO: 643) NO: 645) ID NO: 647) B8 DIQMTQSP RASQRI WYQQKP AASSL EVPSRFSGSQQSYRP FGGGTK VK1 SSLSASVG SNYLN GKAPKLL QS (SEQ GSGTDFTLTI PLT (SEQVDIKR DRVTITC (SEQ ID IY (SEQ ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 651)ID NO: 653) TYYC (SEQ NO: 655) NO: 656) NO: 650) NO: 652) ID NO: 654)B8.1 DIQMTQSP RASQRI WYQQKP AASSL EVPSRFSGS QQSYRP FGGGTK VK1 SSLSASVGSNYLN GKAPKLL QS (SEQ GYGTDFTLT PLT (SEQ VDIKR DRVTITC (SEQ ID IY (SEQID ISSLQPEDFA ID (SEQ ID (SEQ ID NO: 658) ID NO: 660) TYYC (SEQ NO: 662)NO: 663) NO: 657) NO: 659) ID NO: 661) B11 DIVMTQSP RASQSI WYQQKP EASSLEGVPSRFSGS QQYDSY FGGGTK VK1 STLSASVG NSWLA GKAPKLL S (SEQ GSGTEFTLTIWLT  VEIKR DRVTVTC (SEQ ID IY (SEQ ID SSLQPDDFA (SEQ ID (SEQ ID (SEQ IDNO: 665) ID NO: 667) TYYC(SEQ NO: 669) NO: 670) NO: 664) NO: 666)ID NO: 668) B11C3 DIQMTQSP RASQG WYQQRP GASSL GVPSRFSGS QQYDSF FGGGTKVK1 SSVSASVG VSRWL EKAPKLL QS (SEQ GSGTDFTLTI PLT (SEQ VEIKR DRVTITCA (SEQ IY (SEQ ID SSLQPEDFA ID (SEQ ID (SEQ ID ID ID NO: 674) TYYC (SEQNO: 676) NO: 677) NO: 671) NO: 672) NO: 673) ID NO: 675) B11E8 EIVLTQSPRASQS WYQQKR GASTRAT GIPARFSGSG QQYDN FGQGTRL VK1 ATLSVSPG VSKFLAGQAPRLLIY (SEQ ID SGTEFALTIS WPIT EIKR ERATLSC (SEQ ID (SEQ ID NO: 681)SLQSEDFAD (SEQ ID (SEQ ID (SEQ ID NO: 679) NO: 680) YYC (SEQ ID NO: 682)NO: 683) NO: 678) NO: 681) B12 DIVMTQSP RASQGI WYQQKP KASSLES GVPSRFSGSLQHNSY FGQGTKL VK1 STLSASVG SSWLA GKAPKLLIY (SEQ ID GSGTEFTLTI PRA EIKRDRVTITC (SEQ ID (SEQ ID NO: 688) SSLQPEDFA (SEQ ID (SEQ ID (SEQ IDNO: 686) NO: 687) TYYC NO: 690) NO: 691) NO: 685) (SEQ ID NO: 689) B12.1AYVLTQP EGNNV WYQQRP DDSDR GIPERFSGSN QVWDSS FGGGTKL VL3 PSVSVAPG GNKNVHGQAPVL PS SGNTATLTI SAQWV TVLG KTAAITC (SEQ ID VVH (SEQ ID NRVEAGDE(SEQ ID (SEQ ID (SEQ ID NO: 693) (SEQ ID NO: 695) ADYYC NO: 697)NO: 698) NO: 692) NO: 694) (SEQ ID NO: 696) B12.2 ESVLTQPP SGSSSN WYQQLPENSKRSS GIPDRFSGSK GTWDSS FGGGTKL VL1 LVSAAPG IGNNYVS GTAPKLLIY (SEQ IDSGTSATLGIT LSAVV TVLK QKVTISC (SEQ ID (SEQ ID NO: 702) GLQTGDEA (SEQ ID(SEQ ID (SEQ ID NO: 700) NO: 701) DYYC NO: 704) NO: 705) NO: 699)(SEQ ID NO: 703) 1B18 DVVMTQS RASQSI WYQQRP AASSL AVPSRFSGS QQSYSTFGQGTK VK1 PSSVSASV SSYLN GKAPKLL QS GSGTDFTLTI PPT WEIKR (SEQ ID(SEQ ID IF (SEQ ID SSLQPEDFA (SEQ ID (SEQ ID NO: 706) NO: 707) (SEQ IDNO: 709) TYYC NO: 711) NO: 712) NO: 708) (SEQ ID NO: 710) 2B18.1DIVMTQSP RASQI WYQQKP KTSSLES GVPSRFSGR QQSYST FGGGTK VK1 SSLSASVG SSYLNGKAPKLL (SEQ ID GSGTDFTLTI PLT VEIKR DRVSISC (SEQ ID IY NO: 716)SSLQPEDFA (SEQ ID (SEQ ID (SEQ ID NO: 714) (SEQ ID TYYC NO: 718)NO: 719) NO: 713) NO: 715) (SEQ ID NO: 717) 1B22 DIQMTQSP RASQSI WYQQKPSASTLQT GVPSRFSGS QQYNSY FGQGTKL VK1 STLSASIG QSWLA GEAPKLL (SEQ IDGSGTDFTLTI PLT EIKR DRVTISC (SEQ ID IY NO: 723) SSLQPEDFA (SEQ ID(SEQ ID (SEQ ID NO: 721) (SEQ ID TYYC NO: 725) NO: 726) NO: 720)NO: 722) (SEQ ID NO: 724) 4B19 DIVLTQSP RASRSI WYQQRP AASSLHN GVPSRFSGSQQAFGF FGQGTK VK1 STLSASVG GWYLN GKAPKLL (SEQ ID GSGTEFTLTI PRT WEIKRDRVTISC (SEQ ID IY NO: 730) SSLQPDDFA (SEQ ID (SEQ ID (SEQ ID NO: 728)(SEQ ID TYYC NO: 732) NO: 733) NO: 727) NO: 729) (SEQ ID NO: 731) 1B10EIVLTQSP RASQSI WYQQKP KASSLEN GVPSRFSGS QQYSTY FGQGTK VK1 SSLSASVGSSWLA GKAPMV (SEQ ID GSGTEFTLTI SRT VEIKR DRITITC (SEQ ID LIY NO: 737)SSLQPDDFA (SEQ ID (SEQ ID (SEQ ID NO: 735) (SEQ ID TYYC NO: 739)NO: 740) NO: 734) NO: 736) (SEQ ID NO: 738) 1B10.1 EIVLTQSP RASQGIWYQQKP AASSL GVPSRFSGS QQYSSL FGQGTK VK1 SFVSASVG SSWLA GKAPKLL QSGSGTEFTLTI YT VDIKR DRVTITC (SEQ ID IY (SEQ ID SSLQPDDFA (SEQ ID (SEQ ID(SEQ ID NO: 742) (SEQ ID NO: 744) TYYC NO: 746) NO: 747) NO: 741)NO: 743) (SEQ ID NO: 745) 2B18.2 DIVMTQSP RASQSI WYQQKP KTSSLE GVPSRFSGRQQSYST FGGGTK VK1 SSLSASVG SSYLN GKAPKLL S (SEQ GSGTDFTLTI PLT (SEQVEIKR DRVSISC (SEQ ID IY (SEQ ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 749)ID NO: 751) TYYC (SEQ NO: 753) NO: 754) NO: 748) NO: 750) ID NO: 752)2B18.3 DIVMTQSP RASQSS WYQQKP KTSSLE GVPSRFSGS QQSWT FGQGTK VK1 STLSASVGTYWLS GKAPKLL S (SEQ GSGTEFTLTI (SEQ ID VEIKR DRVTITC (SEQ ID IY (SEQ IDSSLQPDDFA NO: 760) (SEQ ID (SEQ ID NO: 756) ID NO: 758) TYYC (SEQNO: 761) NO: 755) NO: 757) ID NO: 759) 1B22.4 DVVMTQS RASQGI WYQQKPDASRL GVPSRFSGS QQYNSY FGGGTKL VK1 PSSVSASV GYWLA GKGPKLL QG GSGTDFTLTIPLT (SEQ EIKR GDRVTITC (SEQ ID IY (SEQ (SEQ ID SSLQPEDFA ID (SEQ ID(SEQ ID NO: 763) ID NO: 765) TYYC (SEQ NO: 767) NO: 768) NO: 762)NO: 764) ID NO: 766) 2B23 DIQMTQSP RTSQGF WYQQKP DASKL GVPSRFSGS QQSNSYFGGGTK VK1 PSLSASVG TSALA GEPPKLLI ES (SEQ GSGTNFALT PLT (SEQ VEIKRDRVTITC (SEQ ID Y (SEQ ID ID ISSLQPEDFA ID (SEQ ID (SEQ ID NO: 770)NO: 771) NO: 772) TYFC (SEQ NO: 774) NO: 775) NO: 769) ID NO: 773) 2B24QSVVTQPP SGSSSN WYQQLP DNNKR GIPDRFSGSK GTWDSS FGGGTKL VL1 SVSAAPG IGNNYGTAPKLL PS (SEQ SGTSATLGIT LSAGV TVLG QKVTISC VS (SEQ IY (SEQ IDGLQTGDEA (SEQ ID (SEQ ID (SEQ ID ID ID NO: 779) DYYC (SEQ NO: 781)NO: 782) NO: 776) NO: 777) NO: 778) ID NO: 780) 2B25 QPGLTQPP SGSSSNWYQQLP RNDQR GVPDRFSGS AAWDD FGGGTQL VL1 SASGTPGQ VGSNT GTAPKLL PS (SEQKSGASASLA SLNGLL TVLG RVTISC VN IY (SEQ ID ISGLRSEDEA (SEQ ID (SEQ ID(SEQ ID (SEQ ID ID NO: 786) DYYC (SEQ NO: 788) NO: 789) NO: 783)NO: 784) NO: 785) ID NO: 787) 2B25.1 ESVLTQPP SGSSSN WYQQLP DNNRRGIPDRFSGSK GTWDSS FGGGTK VL1 SVSAAPG IGNNY GTAPKLL PS (SEQ SGTSATLGITLSEVV VTVLG QKVTISC VS (SEQ IY (SEQ ID GLQTGDEA (SEQ ID (SEQ ID (SEQ IDID ID NO: 793) DYYC (SEQ NO: 795) NO: 796) NO: 790) NO: 791) NO: 792)ID NO: 794) 2B26 QPGLTQPP SGSSSN WYQHLP SNNQR GVPDRFSGS AAWAD FGGGTK VL1SASGTPGQ IGSNPV GTAPKLL PS (SEQ KSGTSASLAI SLNGVV VTVLG RVTISC N (SEQIY (SEQ ID SGLQSEDEA (SEQ ID (SEQ ID (SEQ ID ID ID NO: 800) DYYC (SEQNO: 802) NO: 803) NO: 797) NO: 798) NO: 799) ID NO: 801) 2B27 QSVVTQPPSGSSSN WYQQLP GNNNR GVPDRFSGS QSYDSS FGTGTKL VL1 SVSGAPG IGAGY GTAPKLLPS (SEQ KSGTSASLAI LSAYV TVLG QRVTISC DVH IY (SEQ ID TGLQAEDEA (SEQ ID(SEQ ID (SEQ ID (SEQ ID ID NO: 807) DYYC (SEQ NO: 809) NO: 810) NO: 804)NO: 805) NO: 806) ID NO: 808) 2B28 QSVLTQPP SGSSSN WYQQLP DNNKRGIPDRFSGSK GTWDSS FGGGTQL VL1 SVSAAPG IGNNY GTAPKLL PS (SEQ SGTSATLGITLSAVV TVLG QKVTISC VS (SEQ IY (SEQ ID GLQTGDEA (SEQ ID (SEQ ID (SEQ IDID ID NO: 814) DYYC (SEQ NO: 816) NO: 817) NO: 811) NO: 812) NO: 813)ID NO: 815) 2B29 QPVLTQSP SGSSSN WYQQLP SNNQR GVPDRFSGS ATWDDS FGGGTKLVL1 SASGTPGQ LGSNT GTAPKLL PS (SEQ KSGTSASLAI LSGGV TVLG RVTISC VS (SEQIY (SEQ ID SGLQSEDEA (SEQ ID (SEQ ID (SEQ ID ID ID NO: 821) DYYC (SEQNO: 823) NO: 824) NO: 818) NO: 819) NO: 820) ID NO: 822) 2B30 SYELMQLSGSSSN WYQQLP SNNHR GVPDRFSGS AAWDG FGGGTKL VL1 PSASGTPG IGSNPV GTAPKLLPS (SEQ KSGTSASLAI SLNGHV TVLG QRVSISC N (SEQ IY (SEQ ID SGLQSEDEAV (SEQ (SEQ ID (SEQ ID ID ID NO: 828) DYYC (SEQ ID NO: 831) NO: 825)NO: 826) NO: 827) ID NO: 829) NO: 830) 4B17.1 DIVMTQSP RASQSI WYQQKPKASSL GAPSRFSGS QQSYSIP FGGGTK VK1 SSLSASVG RHYVN GKAPKLL AS (SEQGSGTDFTLTI LT (SEQ VEIKR DRVTISC (SEQ ID IY (SEQ ID SSLQPDDFA ID (SEQ ID(SEQ ID NO: 833) ID NO: 835) TYYC (SEQ NO: 837) NO: 838) NO: 832)NO: 834) ID NO: 836) 4B17.1C DIVMTQSP RASQSI WYQQKP KASSL GAPSRFSGSQQSYSIP FGGGTK VK1 SSLSASVG RHYVN GKAPKLL AS (SEQ GSGTDFTLTI LT (SEQVEIKR DRVTISC (SEQ ID IY (SEQ ID SSLQPDDFA ID (SEQ ID (SEQ ID NO: 840)ID NO: 842) TYYC (SEQ NO: 844) NO: 845) NO: 839) NO: 841) ID NO: 843)4B17.1D DIVMTQSP RASQSI WYQQKP KASSL GAPSRFSGS QQSYSIP FGGGTK VK1SSLSASVG RHYVN GKAPKLL AS (SEQ GSGTDFTLTI LT (SEQ VEIKR DRVTISC (SEQ IDIY (SEQ ID SSLQPDDFA ID (SEQ ID (SEQ ID NO: 847) ID NO: 849) TYYC (SEQNO: 851) NO: 852) NO: 846) NO: 848) ID NO: 850) 4B17.1F DIVMTQSP RASQSIWYQQKP KASSL GAPSRFSGS QQSYSIP FGGGTK VK1 SSLSASVG RHYVN GKAPKLL AS (SEQGSGTDFTLTI LT (SEQ VEIKR DRVTISC (SEQ ID IY (SEQ ID SSLQPDDFA ID (SEQ ID(SEQ ID NO: 854) ID NO: 856) TYYC (SEQ NO: 858) NO: 859) NO: 853)NO: 855) ID NO: 857) 4B17.1G DIVMTQSP RASQSI WYQQKP KASSL GAPSRFSGSQQSYSIP FGGGTK VK1 SSLSASVG RHYVN GKAPKLL AS (SEQ GSGTDFTLTI LT (SEQVEIKR DRVTISC (SEQ ID IY (SEQ ID SSLQPDDFA ID (SEQ ID (SEQ ID NO: 861)ID NO: 863) TYYC (SEQ NO: 865) NO: 866) NO: 860) NO: 862) ID NO: 864)

TABLE 5  BoNT/A light chain antibodies.  Clone VH CDR3 VL CDR3 KD (nM)Epitope ING2 DPYYYSYMDV QQYYSTPFT 0.25 1 (SEQ ID NO: 867) (SEQ IDNO: 868) 5A20.4 EASFGWSYLGHDDAF QQYGSSLWT 0.34 2 DI (SEQ ID(SEQ ID NO: 869) NO: 870) CON1 DPGWIYSDTSAAGWF QQSYDTPRT 10 3 (4A1.1) DP(SEQ ID  (SEQ ID NO: 871) NO: 872) Shown are the clone name, VH CDR3, VLCDR3, K_(D) for BoNT/A light chain, and epitope recognized. Epitopes areassigned sequential numbers, if the epitope does not overlap with otherlight chain antibodies. Affinities are for BoNT/A1 as determined usingyeast displayed scFv and soluble BoNT/A1.

It will be appreciated that the amino acid sequence of a CDR can also bedefined using alternative systems, which will be readily apparent to andapplied by the ordinarily skilled artisan (see, “Sequences of Proteinsof Immunological Interest,” E. Kabat et al., U.S. Department of Healthand Human Services, (1991 and Lefranc et al. IMGT, the internationalImMunoGeneTics information system. Nucl. Acids Res., 2005, 33,D593-D597)). A detailed discussion of the IMGTS system, including howthe IMGTS system was formulated and how it compares to other systems, isprovided on the World Wide Web atimgt.cines.fr/textes/IMGTScientificChart/Numbering/IMGTnumberingsTable.html.An example of a definition of CDRs of an exemplary antibody, 1B10.1, isprovided in FIG. 12. Amino acids indicated in bold are CDR regionsaccording to IMGT boundary definitions, with underlined residues definedaccording to The Kabat system that resulted in the CDRs described above.All amino acid sequences of CDR in the present disclosure are definedaccording to Kabat et al., supra, unless otherwise indicated.

Using the teachings and the sequence information provided herein, thevariable light and variable heavy chains can be joined directly orthrough a linker (e.g., (Gly₄Ser)₃, SEQ ID NO:1) to form a single-chainFv antibody. The various CDRs and/or framework regions can be used toform human antibodies, chimeric antibodies, antibody fragments,polyvalent antibodies, and the like.

Anti-BoNT antibodies of the present disclosure have a binding affinity(K_(D)) for a BoNT protein of at least 10⁻⁸, at least 10⁻⁹, at least10⁻¹⁰, and most preferably at least 10⁻¹¹, 10⁻¹²M or less. Someexemplary K_(D)s (M⁻¹) for BoNT/B or BoNT/E fall in the followingranges: between 5×10⁻¹¹ to 3×10⁻¹⁰, between 4×10⁻¹⁰ to 2×10⁻¹⁰, between7×10⁻¹⁰ to 1×10⁻⁹, between 8×10⁻¹⁰ to 5×10⁻⁹, between 1×10⁻⁹ to 3×10⁻⁹,between 4×10⁻⁹ to 2×10⁻⁸.

The antibody of the present disclosure may be defined by the epitope ofBoNT bound by the antibody. The antibodies provided here may encompassthose that bind to one or more epitopes of BoNT to which an antibodycontaining one or more of the CDRs set forth in Tables 1-5 bind.Epitopes bound by an antibody may be described by a specific BoNT domainand/or the residues therein that contribute to the interaction betweenthe antibody and a BoNT protein. Certain residues of the epitopes boundby the exlempary antibodies are provided in the table below.

TABLE 6 Epitope Data IgG Toxin/Domain Residues Defining Epitope 4E17.1A1/H_(N) Y753 ,E756, E757 1B18 A1/H_(N) Y750, N751, T754 HuC25 A1/H_(C)E920, F953, H1064 AR2/CR1 A1/H_(C) B918, L919, E920, F953, D1062, T1063,H1064 3D12/RAZ1 A1/H_(C) G1129, I1130, R1131 1B11 B1/H_(N) I549 , S5654E17.1 B1/H_(N) K747, R749 , Y750, N751 1B18 B1/H_(N) N751, Y750, Y7533E1 E1/L_(C) N14 , D15 , R16, Q29, E30, Y32 , E135 , K137 , F138 , N140, S142, Q143 , D144 , I145 3E3 E1/L_(C) N14 , Y32, E135 , K137 , F138 ,N140, S142, Q143 , D144 , I145 3E5 E1/L_(C) N14 , D15, Y30 , D144 3E6.1E1/L_(C) S604 , Q607 , Q608 4E11 E1/L_(C) N14 , D15, E30, Y32 4E16.1E1/L_(C) N14 , D15 , E30, Y32 , D144 4E17.1 E1/H_(C) E754b , E755

Numbering system used for the toxin in the table above is based on Lacyet al. (1999) J. Mol. Biol. 291:1091-1104. Residues that are bolded andunderlined have important contributions energetically and eliminationsof these residues often lead to total loss of detectable binding by thecorresponding antibodies to a particular BoNT. Based on the table above,an antibody such as 4E17.1, may be described by its affinity to theH_(N) domain of BoNT/A. The antibody having the same epitope as 4E17.1may also be characterized as an antibody with an epitope(s) containingone or more of the residues: Y753, E756, E757 in BoNT/A. In anotherexample, an antibody such as 3E6.1, may be described by its affinity tothe L_(C) domain of BoNT/E. The antibody having the same epitope as3E6.1 may be characterized as an antibody with an epitope(s) containingone or more of the residues: S604, Q607, Q608 in BoNT/E

The ability of a particular antibody to recognize the same epitope asanother antibody can be determined by the ability of one antibody tocompetitively inhibit binding of the second antibody to the antigen.Competitive inhibition of binding may also be referred to ascross-reactivity of antibodies. Any of a number of competitive bindingassays can be used to measure competition between two antibodies to thesame antigen. For example, a sandwich ELISA assay can be used for thispurpose. Additional methods for assaying for cross-reactivity aredescribed later below.

A first antibody is considered to competitively inhibit binding of asecond antibody, if binding of the second antibody to the antigen isreduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays used to assess competitive binding.

Accordingly, antibodies provided by the present disclosure encompassthose that compete for binding to a BoNT/E with an antibody thatincludes one or more of the V_(H) CDRs set forth in Table 1 and/or oneor more of the V_(L) CDRs set forth in Table 2. Antibodies provided bythe present disclosure also encompass those that compete for binding toa BoNT/B with an antibody that includes one or more of the V_(H) CDRsset forth in Table 3 and/or one or more of the V_(L) CDRs set forth inTable 4. Additional antibodies may encompass those that compete forbinding to a BoNT/A with an antibody with one or more CDRs set forth inTable 5.

For example, an antibody may have the binding specificity (i.e., in thiscontext, the same CDRs, or substantially the same CDRs) of an antibodyhaving V_(H) and V_(L) CDRs or full length V_(H) and V_(L) as set forthin Tables 1-5. An antibody of the present disclosure may thereforecontain a CDR as set forth in a V_(H) or V_(L) sequence shown in Tables1-5 and, additionally, may have at least 80% identity, preferably, 85%,90%, or 95% identity to a full-length V_(H) or V_(L) sequence. Forexample, an antibody may contain the CDRs of a V_(H) and a V_(L)sequence and human framework sequences set forth in Tables 1-5.

III. Preparation of BoNT Neutralizing Antibodies.

A) Recombinant Expression of BoNT-Neutralizing Antibodies.

Using the information provided herein, the botulinumneurotoxin-neutralizing antibodies of the present disclosure areprepared using standard techniques well known to those of skill in theart.

For example, the polypeptide sequences provided herein (see, e.g.,Tables 1-5, and/or FIGS. 1-2 and 11) can be used to determineappropriate nucleic acid sequences encoding the BoNT-neutralizingantibodies and the nucleic acids sequences then used to express one ormore BoNT-neutralizing antibodies. The nucleic acid sequence(s) can beoptimized to reflect particular codon “preferences” for variousexpression systems according to standard methods well known to those ofskill in the art.

Using the sequence information provided, the nucleic acids may besynthesized according to a number of standard methods known to those ofskill in the art. Oligonucleotide synthesis, is preferably carried outon commercially available solid phase oligonucleotide synthesis machines(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) ormanually synthesized using, for example, the solid phase phosphoramiditetriester method described by Beaucage et. al. (1981) Tetrahedron Letts.22(20): 1859-1862.

Once a nucleic acid encoding an anti-BoNT antibody is synthesized it canbe amplified and/or cloned according to standard methods. Molecularcloning techniques to achieve these ends are known in the art. A widevariety of cloning and in vitro amplification methods suitable for theconstruction of recombinant nucleic acids are known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook);and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methodsof producing recombinant immunoglobulins are also known in the art. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Qβ-replicase amplification andother RNA polymerase mediated techniques are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Amheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080;Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene4, 560; and Barringer et al. (1990) Gene 89, 117. Improved methods ofcloning in vitro amplified nucleic acids are described in Wallace etal., U.S. Pat. No. 5,426,039.

Once the nucleic acid for an anti-BoNT antibody is isolated and cloned,one can express the gene in a variety of recombinantly engineered cellsknown to those of skill in the art. Examples of such cells includebacteria, yeast, filamentous fungi, insect (especially employingbaculoviral vectors), and mammalian cells. It is expected that those ofskill in the art are knowledgeable in the numerous expression systemsavailable for expression of antibodies.

In brief summary, the expression of natural or synthetic nucleic acidsencoding anti-BoNT antibodies will typically be achieved by operablylinking a nucleic acid encoding the antibody to a promoter (which iseither constitutive or inducible), and incorporating the construct intoan expression vector. The vectors can be suitable for replication andintegration in prokaryotes, eukaryotes, or both. Typical cloning vectorscontain transcription and translation terminators, initiation sequences,and promoters useful for regulation of the expression of the nucleicacid encoding the anti-BoNT antibody. The vectors optionally comprisegeneric expression cassettes containing at least one independentterminator sequence, sequences permitting replication of the cassette inboth eukaryotes and prokaryotes, i.e., shuttle vectors, and selectionmarkers for both prokaryotic and eukaryotic systems. See Sambrook et al(1989) supra.

To obtain high levels of expression of a cloned nucleic acid it iscommon to construct expression plasmids which typically contain a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.

Examples of regulatory regions suitable for this purpose in E. coli arethe promoter and operator region of the E. coli tryptophan biosyntheticpathway as described by Yanofsky (1984) J. Bacteriol., 158:1018-1024,and the leftward promoter of phage lambda (P_(L)) as described byHerskowitz and Hagen (1980) Ann. Rev. Genet., 14:399-445 and theL-arabinose (araBAD) operon (Better (1999) Gene Exp Systems pp 95-107Academic Press, Inc., San Diego, Calif.). The inclusion of selectionmarkers in DNA vectors transformed in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol. See Sambrook et al (1989) supra fordetails concerning selection markers, e.g., for use in E. coli.

Expression systems for expressing anti-BoNT antibodies are availableusing, for example, E. coli, Bacillus sp. (see, e.g., Palva, et al.(1983) Gene 22:229-235; Mosbach et al. (1983) Nature, 302: 543-545), andSalmonella. E. coli systems may also be used.

The anti-BoNT antibodies produced by prokaryotic cells may requireexposure to chaotropic agents for proper folding. During purificationfrom, e.g., E. coli, the expressed protein is optionally denatured andthen renatured. This can be accomplished, e.g., by solubilizing thebacterially produced antibodies in a chaotropic agent such as guanidineHCl. The antibody is then renatured, either by slow dialysis or by gelfiltration (see, e.g., U.S. Pat. No. 4,511,503). Alternatively, nucleicacid encoding the anti-BoNT antibodies may be operably linked to asecretion signal sequence such as pelB so that the anti-BoNT antibodiesare secreted into the medium in correctly-folded form (Better et al(1988) Science 240: 1041-1043).

Methods of transfecting and expressing genes in mammalian cells areknown in the art (see e.g. Birch and Racher Adv. Drug Deliv. Rev. 2006,58: 671-685). Transducing cells with nucleic acids can involve, forexample, incubating viral vectors containing anti-BoNT nucleic acidswith cells within the host range of the vector (see, e.g., Goeddel(1990) Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego,Calif. or Krieger (1990) Gene Transfer and Expression—A LaboratoryManual, Stockton Press, New York, N.Y. and the references citedtherein).

The culture of cells used in the present disclosure, including celllines and cultured cells from tissue or blood samples is well known inthe art (see, e.g., Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, third edition, Wiley-Liss, N.Y. and the referencescited therein).

Techniques for using and manipulating antibodies are found in Coligan(1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature256: 495-497.

The BoNT-neutralizing antibody gene(s) (e.g. BoNT-neutralizing scFvgene) may be subcloned into the expression vector pUC119mycHis(Tomlinson et al. (1996) J. Mol. Biol., 256: 813-817) or pSYN3,resulting in the addition of a hexahistidine tag at the C-terminal endof the scFv to facilitate purification. Detailed protocols for thecloning and purification of certain BoNT-neutralizing antibodies arefound, for example, in Amersdorfer et al. (1997) Infect. Immunity,65(9): 3743-3752, and the like.

B) Preparation of Whole Polyclonal or Monoclonal Antibodies.

The anti-BoNT antibodies of the present disclosure include individual,allelic, strain, or species variants, and fragments thereof, both intheir naturally occurring (full-length) forms and in recombinant forms.Certain antibodies may be selected to bind one or more epitopes bound bythe antibodies described herein (e.g., 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1,3E5, 3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, A12,6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2,1B18, 2B18.1, 4B19, 4B19.1, 1B22, 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4,2B23, 2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2,B11.A5, B11.F7, B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1,4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2). Theantibodies can be raised in their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated. Manymethods of making antibodies that specifically bind to a particularepitope are known to persons of skill The following discussion ispresented as a general overview of the techniques available; however,one of skill will recognize that many variations upon the followingmethods are known.

1) Polyclonal Antibody Production.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen (e.g., BoNT/A, BoNT/B, BoNT/E, etc.),subsequences including, but not limited to subsequences comprisingepitopes specifically bound by antibodies expressed by clones clones2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 3E6.2, 4E11, 4E13,4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11,B11C3, B11E8, B12, B12.1, B12.2, 1B18, 2B18.1, 4B19, 4B19.1, 1B22, 1B10,1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23, 2B24, 2B25, 2B25.1, 2B26, 2B27,2B28, 2B29, 2B30, 4B17.1, 4B17.1C, 4B17.1D, 4B17.1F, 4B17.1G, 3E6.2,4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2, B11.A5, B11.F7, B11.H12, B11.E9,4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1,ING1.2B10, and ING1.3C2 disclosed herein, preferably a purifiedpolypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST,keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated intoan immunization vector such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the polypeptide of interest. When appropriately hightiters of antibody to the immunogen are obtained, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the BoNT polypeptide isperformed where desired (see, e.g., Coligan (1991) Current Protocols inImmunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: ALaboratory Manual Cold Spring Harbor Press, NY).

Antibodies that specifically bind to the neutralizing epitopes describedherein can be selected from polyclonal sera using the selectiontechniques described herein.

2) Monoclonal Antibody Production.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Descriptions of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497.

Summarized briefly, monoclonal antibody production using hybridomas mayproceed by injecting an animal with an (e.g., BoNT/A, BoNT/B, BoNT/E,etc.) subsequences including, but not limited to subsequences comprisingepitopes specifically bound by antibodies expressed by clones 2A10, 3E1,3E2, 3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1,4E17, 4E17.1, A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8,B12, B12.1, B12.2, 1B18, 2B18.1, 4B19, 4B19.1, 1B22, 1B10, 1B10.1,2B18.2, 2B18.3, 1B22.4, 2B23, 2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28,2B29, 2B30, 4B17.1, 4B17.1C, 4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4,4E17.6, 4B19.1, B6.C12, B6.D2, B11.A5, B11.F7, B11.H12, B11.E9, 4B1,4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1,ING1.2B10, and ING1.3C2 disclosed herein. The animal is then sacrificedand cells taken from its spleen, which are fused with myeloma cells. Theresult is a hybrid cell or “hybridoma” that is capable of reproducingantibodies in vitro. The population of hybridomas is then screened toisolate individual clones, each of which secretes a single antibodyspecies to the immunogen. In this manner, the individual antibodyspecies obtained are the products of immortalized and cloned single Bcells from the immune animal generated in response to a specific siterecognized on the immunogenic substance.

Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe BoNT antigen, and yield of the monoclonal antibodies produced bysuch cells is enhanced by various techniques, including injection intothe peritoneal cavity of a vertebrate (preferably mammalian) host. Theantibodies of the present disclosure are used with or withoutmodification, and include chimeric antibodies such as humanized murineantibodies.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of hybridomasare contemplated for the present BoNT (e.g., BoNT/B) binding antibodiesand fragments. DNA is cloned into a bacterial expression system. Oneexample of a suitable technique uses a bacteriophage lambda vectorsystem having a leader sequence that causes the expressed Fab protein tomigrate to the periplasmic space (between the bacterial cell membraneand the cell wall) or to be secreted. One can rapidly generate andscreen great numbers of functional Fab fragments for those which bindBoNT. Such BoNT binding agents (Fab fragments with specificity for aBoNT polypeptide) are specifically encompassed within the BoNT bindingantibodies and fragments of the present disclosure. Other methods forscreening and production of antibodies may employ one or more of displaysystems such as phage display, yeast display, ribosome, etc., and anantibody production system such as that derived from transgenic mice.

IV. Modification of BoNT Neutralizing Antibodies.

A) Display Techniques can be used to Increase Antibody Affinity.

To create higher affinity antibodies, mutant scFv gene repertories,based on the sequence of a binding scFv (see, e.g., Tables 1-5, and/orFIGS. 1, 2, and/or 11), can be created and expressed on the surface ofphage. Display of antibody fragments on the surface of viruses whichinfect bacteria (bacteriophage or phage) makes it possible to producehuman or other mammalian antibodies (e.g., scFvs) with a wide range ofaffinities and kinetic characteristics. To display antibody fragments onthe surface of phage (phage display), an antibody fragment gene isinserted into the gene encoding a phage surface protein (e.g., pIII) andthe antibody fragment-pIII fusion protein is expressed on the phagesurface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom etal. (1991) Nucleic Acids Res., 19: 4133-4137).

Since the antibody fragments on the surface of the phage are functional,those phage bearing antigen binding antibody fragments can be separatedfrom non-binding or lower affinity phage by antigen affinitychromatography (McCafferty et al. (1990) Nature, 348: 552-554). Mixturesof phage are allowed to bind to the affinity matrix, non-binding orlower affinity phage are removed by washing, and bound phage are elutedby treatment with acid or alkali.

By infecting bacteria with the eluted phage or modified variants of theeluted phage as described below, more phage can be grown and subjectedto another round of selection. In this way, an enrichment of 1000 foldin one round may become 1,000,000 fold in two rounds of selection (see,e.g., McCafferty et al. (1990) Nature, 348: 552-554). Thus, even whenenrichments in each round are low, multiple rounds of affinity selectionleads to the isolation of rare phage and the genetic material containedwithin which encodes the sequence of the binding antibody (see, e.g.,Marks et al. (1991) J. Mol. Biol., 222: 581-597). The physical linkbetween genotype and phenotype provided by phage display makes itpossible to test every member of an antibody fragment library forbinding to antigen, even with libraries as large as 100,000,000 clones.For example, after multiple rounds of selection on antigen, a bindingscFv that occurred with a frequency of only 1/30,000,000 clones wasrecovered (Marks et al. (1991) J. Mol. Biol., 222: 581-597.).

Yeast display may also be utilized to increase antibody affinity and hasthe ability to finely discriminate between mutants of close affinity.Antibody variable region genes (V-genes) may be diversified eitherrandomly or using spiked oligonucleotides, and higher affinity mutantsselected using various types of affinity chromatography or flowcytometry.

1) Chain Shuffling.

One approach for creating mutant scFv gene repertoires involvesreplacing either the V_(H) or V_(L) gene from a binding scFv with arepertoire of V_(H) or V_(L) genes (chain shuffling) (see, e.g.,Clackson et al. (1991) Nature, 352: 624-628). Such gene repertoirescontain numerous variable genes derived from the same germline gene asthe binding scFv, but with point mutations (see, e.g., Marks et al.(1992) Bio/Technology, 10: 779-783). Using light or heavy chainshuffling and phage display, the binding avidities of, e.g., BoNT/E orBoNT/B-neutralizing antibody fragment can be dramatically increased(see, e.g., Marks et al. (1992) Bio/Technology, 10: 779-785 in which theaffinity of a human scFv antibody fragment which bound the haptenphenyloxazolone (phox) was increased from 300 nM to 15 nM (20 fold)).

Thus, to alter the affinity of BoNT-neutralizing antibody a mutant scFvgene repertoire may be created containing the V_(H) gene of a knownBoNT-neutralizing antibody (e.g., 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5,3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12,B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18,2B18.1, 4B19, 4B19.1, 1B22, 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23,2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4,4E17.6, 4B19.1, B6.C12, B6.D2,B11.A5, B11.F7, B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1,4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2) and a V_(L)gene repertoire (light chain shuffling). Alternatively, an scFv generepertoire is created containing the V_(L) gene of a knownBoNT-neutralizing antibody (e.g 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5,3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12,B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18,2B18.1, 4B19, 4B19.1, 1B22, 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23,2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2,B11.A5, B11.F7, B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1,4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2) and a V_(H)gene repertoire (heavy chain shuffling). The scFv gene repertoire may becloned into a phage display vector (e.g., pHEN-1, Hoogenboom et al.(1991) Nucleic Acids Res., 19: 4133-4137) and after transformation alibrary of transformants is obtained. Phage are prepared andconcentrated and selections are performed. In addition to chainshuffling, it is also possible to shuffle individual complementaritydetermining regions (CDRs).

The antigen concentration may be decreased in each round of selection,reaching a concentration less than the desired K_(d) by the final roundsof selection. This results in the selection of phage on the basis ofaffinity (Hawkins et al. (1992) J. Mol. Biol. 226: 889-896).

Chain shuffling may be combined with the stringent selections madepossible by yeast display and flow cytometry. This novel approach wasfound to be particularly powerful for increasing antibody affinity (seeexample 1).

2) Increasing the Affinity of Anti-BoNT Antibodies by Site DirectedMutagenesis.

The majority of antigen contacting amino acid side chains are located inthe complementarity determining regions (CDRs), three in the V_(H)(CDR1, CDR2, and CDR3) and three in the V_(L) (CDR1, CDR2, and CDR3)(see, e.g., Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothiaet al. (1986) Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol.,217: 133-151). Without being bound to a theory, it is believed thatthese residues contribute the majority of binding energetics responsiblefor antibody affinity for antigen. In other molecules, mutating aminoacids that contact ligand has been shown to be an effective means ofincreasing the affinity of one protein molecule for its binding partner(Lowman et al. (1993) J. Mol. Biol., 234: 564-578; Wells (1990)Biochemistry, 29: 8509-8516). Thus mutation (randomization) of the CDRsand screening against, for example, BoNT/A, BoNT/E, BoNT/B, or theepitopes thereof, can be used to generate anti-BoNT antibodies havingimproved binding affinity.

Each CDR is randomized in a separate library, using, for example, A12 asa template. To simplify affinity measurement, A12, or other loweraffinity anti-BoNT antibodies, are used as a template, rather than ahigher affinity scFv. The CDR sequences of the highest affinity mutantsfrom each CDR library are combined to obtain an additive increase inaffinity. A similar approach has been used to increase the affinity ofhuman growth hormone (hGH) for the growth hormone receptor over 1500fold from 3.4×10⁻¹⁰ to 9.0×10⁻¹³ M (see, e.g., Lowman et al. (1993) J.Mol. Biol., 234: 564-578).

To increase the affinity of BoNT-neutralizing antibodies, amino acidresidues located in one or more CDRs (e.g., 9 amino acid residueslocated in V_(L) CDR3) are partially randomized by synthesizing a“doped” oligonucleotide in which the wild type nucleotide occurred witha frequency of, e.g. 49%. The oligonucleotide is used to amplify theremainder of the BoNT-neutralizing scFv gene(s) using PCR.

For example, to create a library in which V_(H) CDR3 is randomized, anoligonucleotide is synthesized which anneals to the BoNT-neutralizingantibody V_(H) framework 3 and encodes V_(H) CDR3 and a portion offramework 4. At the four positions to be randomized, the sequence NNScan be used, where N is any of the 4 nucleotides, and S is “C” or “T”.The oligonucleotide is used to amplify the BoNT-neutralizing antibodyV_(H) gene using PCR, creating a mutant BoNT-neutralizing antibody V_(H)gene repertoire. PCR is used to splice the V_(H) gene repertoire withthe BoNT-neutralizing antibody light chain gene, and the resulting scFvgene repertoire cloned into a phage display vector (e.g., pHEN-1 orpCANTAB5E). Ligated vector DNA is used to transform electrocompetent E.coli to produce a phage antibody library.

To select higher affinity mutant scFv, each round of selection of thephage antibody libraries is conducted on decreasing amounts of one ormore BoNT subtypes. Clones from the third and fourth round of selectioncan screened for binding to the desired antigen(s) (e.g., BoNT/B BoNT/E,etc.) by ELISA on 96 well plates. scFv from, e.g., twenty to forty ELISApositive clones can be expressed, e.g. in 10 ml cultures, the periplasmharvested, and the scFv k_(off) determined by BIAcore. Clones with theslowest k_(off) are sequenced, and each unique scFv subcloned into anappropriate vector (e.g., pUC119 mycHis). The scFv are expressed inculture, and purified. Affinities of purified scFv can be determined byBIAcore.

By way of illustration, FIG. 5 shows a scheme used for affinitymaturation of HuC25 (FIG. 5A) and 3D12 (FIG. 5B) scFv using yeastdisplay (see, e.g., Ser. No. 11/342,271, filed on Jan. 26, 2006, Ser.No. 09/144,886, filed on Aug. 31, 1998, Ser. No. 10/632,706, filed onAug. 1, 2003, U.S. Ser. No. 60/942,173 filed on Jun. 5, 2008, and PCTapplication Nos: PCT/US2006/003070 and PCT/US03/24371, which areincorporated herein by reference for all purposes).

3) Creation of Anti-BoNT (scFv′)2 Homodimers.

To create anti-BoNT (e.g., BoNT-neutralizing) (scFv′)₂ antibodies, twoanti-BoNT scFvs are joined, either through a linker (e.g., a carbonlinker, a peptide, etc.) or through a disulfide bond between, forexample, two cysteines. Thus, for example, to create disulfide linkedscFv, a cysteine residue can be introduced by site directed mutagenesisbetween a myc tag and a hexahistidine tag at the carboxy-terminus of ananti-BoNT/B. Introduction of the correct sequence can be verified by DNAsequencing. The construct may be in pUC119, so that the pelB leaderdirects expressed scFv to the periplasm and cloning sites (NcoI andNotI) exist to introduce anti-BoNT mutant scFv. Expressed scFv has themyc tag at the C-terminus, followed by two glycines, a cysteine, andthen 6 histidines to facilitate purification by IMAC. After disulfidebond formation between the two cysteine residues, the two scFv can beseparated from each other by 26 amino acids (two 11 amino acid myc tagsand 4 glycines). An scFv expressed from this construct, purified by IMACmay predominantly comprise monomeric scFv. To produce (scFv′)₂ dimers,the cysteine can be reduced by incubation with 1 MMbeta-mercaptoethanol, and half of the scFv blocked by the addition ofDTNB. Blocked and unblocked scFvs can be incubated together to form(scFv′)₂ and the resulting material can optionally be analyzed by gelfiltration. The affinity of the anti-BoNT scFv′ monomer and (scFv′)₂dimer can optionally be determined by BIAcore.

The (scFv′)₂ dimer may be created by joining the scFv fragments througha linker, more preferably through a peptide linker. This can beaccomplished by a wide variety of means well known to those of skill inthe art. For example, one preferred approach is described by Holliger etal. (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (see also WO94/13804).

Typically, linkers are introduced by PCR cloning. For example, syntheticoligonucleotides encoding the 5 amino acid linker (Gly₄Ser, SEQ IDNO:938) can be used to PCR amplify the BoNT-neutralizing antibody V_(H)and V_(L) genes which are then spliced together to create theBoNT-neutralizing diabody gene. The gene can then be cloned into anappropriate vector, expressed, and purified according to standardmethods well known to those of skill in the art.

4) Preparation of Anti-BoNT (scFv)₂, Fab, and (Fab′)₂ Molecules.

Anti-BoNT antibodies such as anti-BoNT/E or anti-BoNT/B scFv, orvariant(s) with higher affinity, are suitable templates for creatingsize and valency variants. For example, an anti-BoNT (scFv′)₂ can becreated from the parent scFv (e.g., 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1,3E5, 3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12,6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2,1B18, 2B18.1, 4B19, 4B19.1, 1B22, 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4,2B23, 2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2,B11.A5, B11.F7, B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1,4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2, etc.) asdescribed above. An scFv gene can be excised using appropriaterestriction enzymes and cloned into another vector as described herein.

Expressed scFv may include a myc tag at the C-terminus, followed by twoglycines, a cysteine, and six histidines to facilitate purification.After disulfide bond formation between the two cystine residues, the twoscFv may be separated from each other by 26 amino acids (e.g., twoeleven amino acid myc tags and four glycines). Single-chain Fv (scFv)can be expressed from this construct and purified.

To produce (scFv′)₂ dimers, the cysteine is reduced by incubation with 1mM β-mercaptoethanol, and half of the scFv blocked by the addition ofDTNB. Blocked and unblocked scFv are incubated together to form(scFv′)₂, which is purified. As higher affinity scFv are isolated, theirgenes are similarly used to construct (scFv′)₂.

Anti-BoNT Fab may also be expressed in E. coli using an expressionvector similar to the one described by Better et. al. (1988) Science,240: 1041-1043. For example, to create a BoNT/B or BoNT/E-neutralizingFab, the V_(H) and V_(L) genes are amplified from the scFv using PCR.The V_(H) gene is cloned into an expression vector (e.g., a pUC119 basedbacterial expression vector) that provides an IgG C_(H)1 domaindownstream from, and in frame with, the V_(H) gene. The vector alsocontains the lac promoter, a pelB leader sequence to direct expressedV_(H)-C_(H)1 domain into the periplasm, a gene 3 leader sequence todirect expressed light chain into the periplasm, and cloning sites forthe light chain gene. Clones containing the correct V_(H) gene areidentified, e.g., by PCR fingerprinting. The V_(L) gene is spliced tothe C_(L) gene using PCR and cloned into the vector containing the V_(H)C_(H)1 gene.

B) Selection of Neutralizing Antibodies.

Selection of anti-BoNT antibodies (whether produced by phage display,yeast display, immunization methods, hybridoma technology, etc.)involves screening the resulting antibodies for specific binding to anappropriate antigen(s). In the instant case, suitable antigens caninclude, but are not limited to BoNT/E1, BoNT/E2, BoNT/E3, BoNT/B1,BoNT/B2, BoNT/B3, BoNT/B4, BoNT/A1, BoNT/A2, BoNT/A3, a C-terminaldomain of BoNT heavy chain (binding domain), BoNT holotoxins,recombinant BoNT domains such as H_(C) (binding domain), H_(N)(translocation domain), or L_(C) (light chain), and the like. Theneutralizing antibodies may be selected for specific binding of anepitope recognized by one or more of the antibodies described herein.

Selection can be by any of a number of methods well known to those ofskill in the art. In one example, selection is by immunochromatography(e.g., using immunotubes, Maxisorp, Nunc) against the desired target,e.g., BoNT/E, BoNT/B, etc. In a related example, selection is against aBoNT protein in a surface plasmon resonance system (e.g., BIAcore,Pharmacia) either alone or in combination with an antibody that binds toan epitope specifically bound by one or more of the antibodies describedherein. Selection can also be done using flow cytometry for yeastdisplay libraries. Yeast display libraries are sequentially selected,first on BoNT/B1, then on other BoNT/B subtypes (BoNT/B2, B3 and B4) toobtain antibodies that bind with high affinity to all subtypes ofBoNT/B. This can be repeated for other subtypes.

For phage display, analysis of binding can be simplified by including anamber codon between the antibody fragment gene and gene III. This makesit possible to easily switch between displayed and soluble antibodyfragments simply by changing the host bacterial strain. When phage aregrown in a supE suppresser strain of E. coli, the amber stop codonbetween the antibody gene and gene III is read as glutamine and theantibody fragment is displayed on the surface of the phage. When elutedphage are used to infect a non-suppressor strain, the amber codon isread as a stop codon and soluble antibody is secreted from the bacteriainto the periplasm and culture media (Hoogenboom et al. (1991) NucleicAcids Res., 19: 4133-4137). Binding of soluble scFv to antigen can bedetected, e.g., by ELISA using a murine IgG monoclonal antibody (e.g.,9E10) which recognizes a C-terminal myc peptide tag on the scFv (Evan etal. (1985) Mol. Cell Biol., 5: 3610-3616; Munro et al. (1986) Cell, 46:291-300), e.g., followed by incubation with polyclonal anti-mouse Fcconjugated to a detectable label (e.g., horseradish peroxidase).

As indicated above, purification of the anti-BoNT antibody can befacilitated by cloning of the scFv gene into an expression vector (e.g.,expression vector pUC119mycHIS) that results in the addition of the mycpeptide tag followed by a hexahistidine tag at the C-terminal end of thescFv. The vector also preferably encodes the pectate lyase leadersequence that directs expression of the scFv into the bacterialperiplasm where the leader sequence is cleaved. This makes it possibleto harvest native properly folded scFv directly from the bacterialperiplasm. The BoNT-neutralizing antibody is then expressed and purifiedfrom the bacterial supernatant using immobilized metal affinitychromatography.

C) Measurement of Anti-BoNT Antibody Affinity for One or More BoNTSubtypes.

As explained above, selection for increased avidity involves measuringthe affinity of an anti-BoNT (e.g., a BoNT-neutralizing) antibody (or amodified BoNT-neutralizing antibody) for one or more targets of interest(e.g. BoNT/E subtype(s) or domains thereof. For example, the K_(D) of aBoNT/E-neutralizing antibody and the kinetics of binding to BoNT/E aredetermined in a BIAcore, a biosensor based on surface plasmon resonance.For this technique, antigen is coupled to a derivatized sensor chipcapable of detecting changes in mass. When antibody is passed over thesensor chip, antibody binds to the antigen resulting in an increase inmass that is quantifiable. Measurement of the rate of association as afunction of antibody concentration can be used to calculate theassociation rate constant (k_(on)). After the association phase, bufferis passed over the chip and the rate of dissociation of antibody(k_(off)) determined. K_(on) is typically measured in the range 1.0×10²to 5.0×10⁶ M and k_(off) in the range 1.0×10⁻¹ to 1.0×10⁻⁶M. Theequilibrium constant K_(d) is then calculated as k_(off)/k_(on) and thusis typically measured in the range 10⁻⁵ to 10⁻¹²M. Affinities measuredin this manner correlate well with affinities measured in solution byfluorescence quench titration.

Phage display and selection generally results in the selection of higheraffinity mutant scFvs (Marks et al. (1992) Bio/Technology, 10: 779-783;Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; Riechmann et al.(1993) Biochemistry, 32: 8848-8855; Clackson et al. (1991) Nature, 352:624-628), but probably does not result in the separation of mutants withless than a 6 fold difference in affinity (Riechmann et al. (1993)Biochemistry, 32: 8848-8855). Thus a rapid method is needed to estimatethe relative affinities of mutant scFvs isolated after selection. Sinceincreased affinity results primarily from a reduction in the k_(off),measurement of k_(off) should identify higher affinity scFv. k_(off) canbe measured in the BIAcore on unpurified scFv in bacterial periplasm,since expression levels are high enough to give an adequate bindingsignal and k_(off) is independent of concentration. The value of k_(off)for periplasmic and purified scFv is typically in close agreement.

V. Humanized, Human Engineered or Human Antibody Production.

The present BoNT (e.g., BoNT/B) binding antibodies and fragments can behumanized or human engineered antibodies. As used herein, a humanizedantibody, or antigen binding fragment thereof, is a recombinantpolypeptide that comprises a portion of an antigen binding site from anon-human antibody and a portion of the framework and/or constantregions of a human antibody. A human engineered antibody or antibodyfragment may be derived from a human or non-human (e.g., mouse) sourcethat has been engineered by modifying (e.g., deleting, inserting, orsubstituting) amino acids at specific positions so as to alter certainbiophysical properties or to reduce any detectable immunogenicity of themodified antibody in a human.

Humanized antibodies also encompass chimeric antibodies and CDR-graftedantibodies in which various regions may be derived from differentspecies. Chimeric antibodies may be antibodies that include a non-humanantibody variable region linked to a human constant region. Thus, inchimeric antibodies, the variable region is mostly non-human, and theconstant region is human. Chimeric antibodies and methods for makingthem are described in Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6841-6855 (1984), Boulianne, et al., Nature, 312: 643-646 (1984), andPCT Application Publication WO 86/01533. Although, they can be lessimmunogenic than a mouse monoclonal antibody, administrations ofchimeric antibodies have been associated with human anti-mouse antibodyresponses (HAMA) to the non-human portion of the antibodies. Chimericantibodies can also be produced by splicing the genes from a mouseantibody molecule of appropriate antigen-binding specificity togetherwith genes from a human antibody molecule of appropriate biologicalactivity, such as the ability to activate human complement and mediateADCC. Morrison et al. (1984), Proc. Natl. Acad. Sci., 81: 6851;Neuberger et al. (1984), Nature, 312: 604. One example is thereplacement of an Fc region with that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs from anon-human “donor” antibody linked to the framework region from a human“recipient” antibody. Generally, CDR-grafted antibodies include morehuman antibody sequences than chimeric antibodies because they includeboth constant region sequences and variable region (framework) sequencesfrom human antibodies. Thus, for example, a CDR-grafted humanizedantibody may comprise a heavy chain that comprises a contiguous aminoacid sequence (e.g., about 5 or more, 10 or more, or even 15 or morecontiguous amino acid residues) from the framework region of a humanantibody (e.g., FR-1, FR-2, or FR-3 of a human antibody) or, optionally,most or all of the entire framework region of a human antibody.CDR-grafted antibodies and methods for making them are described in,Jones et al., Nature, 321: 522-525 (1986), Riechmann et al., Nature,332: 323-327 (1988), and Verhoeyen et al., Science, 239: 1534-1536(1988)). Methods that can be used to produce humanized antibodies alsoare described in U.S. Pat. Nos. 4,816,567, 5,721,367, 5,837,243, and6,180,377. CDR-grafted antibodies are considered less likely thanchimeric antibodies to induce an immune reaction against non-humanantibody portions. However, it has been reported that frameworksequences from the donor antibodies are required for the bindingaffinity and/or specificity of the donor antibody, presumably becausethese framework sequences affect the folding of the antigen-bindingportion of the donor antibody. Therefore, when donor, non-human CDRsequences are grafted onto unaltered human framework sequences, theresulting CDR-grafted antibody can exhibit, in some cases, loss ofbinding avidity relative to the original non-human donor antibody. See,e.g., Riechmann et al., Nature, 332: 323-327 (1988), and Verhoeyen etal., Science, 239: 1534-1536 (1988).

Human engineered antibodies include for example “veneered” antibodiesand antibodies prepared using HUMAN ENGINEERING™ technology (U.S. Pat.No. 5,869,619). HUMAN ENGINEERING™ technology is commercially available,and involves altering an non-human antibody or antibody fragment, suchas a mouse or chimeric antibody or antibody fragment, by making specificchanges to the amino acid sequence of the antibody so as to produce amodified antibody with reduced immunogenicity in a human thatnonetheless retains the desirable binding properties of the originalnon-human antibodies. Techniques for making human engineered proteinsare described in Studnicka et al., Protein Engineering, 7: 805-814(1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619,and PCT Application Publication WO 93/11794.

“Veneered” antibodies are non-human or humanized (e.g., chimeric orCDR-grafted antibodies) antibodies that have been engineered to replacecertain solvent-exposed amino acid residues so as to further reducetheir immunogenicity or enhance their function. As surface residues of achimeric antibody are presumed to be less likely to affect properantibody folding and more likely to elicit an immune reaction, veneeringof a chimeric antibody can include, for instance, identifyingsolvent-exposed residues in the non-human framework region of a chimericantibody and replacing at least one of them with the correspondingsurface residues from a human framework region. Veneering can beaccomplished by any suitable engineering technique, including the use ofthe above-described HUMAN ENGINEERING™ technology.

In a different approach, a recovery of binding avidity can be achievedby “de-humanizing” a CDR-grafted antibody. De-humanizing can includerestoring residues from the donor antibody's framework regions to theCDR grafted antibody, thereby restoring proper folding. Similar“de-humanization” can be achieved by (i) including portions of the“donor” framework region in the “recipient” antibody or (ii) graftingportions of the “donor” antibody framework region into the recipientantibody (along with the grafted donor CDRs).

For a further discussion of antibodies, humanized antibodies, humanengineered, and methods for their preparation, see Kontermann and Dubel,eds., Antibody Engineering, Springer, New York, N.Y., 2001.

The present antibodies and fragments encompass human antibodies, such asantibodies which bind BoNT polypeptides and are encoded by nucleic acidsequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence, and fragments, syntheticvariants, derivatives and fusions thereof. Such antibodies may beproduced by any method known in the art, such as through the use oftransgenic mammals (such as transgenic mice) in which the nativeimmunoglobulin repertoire has been replaced with human V-genes in themammal chromosome. Such mammals appear to carry out VDJ recombinationand somatic hypermutation of the human germline antibody genes in anormal fashion, thus producing high affinity antibodies with completelyhuman sequences.

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598, 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. The monoclonalantibodies can be tested for the ability to inhibit or neutralize thebiological activity or physiological effect of the correspondingprotein. Human monoclonal antibodies with specificity for the antigenused to immunize transgenic animals are also disclosed in WO 96/34096and U.S. patent application no. 20030194404; and U.S. patent applicationno. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851, 1996), which containsgene sequences from unrearranged human antibody genes that code for theheavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hlg)loci. The TCMOUSE™ has a fully diverse repertoire of hlgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TC MOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies. See also Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immunol., 7:33 (1993); and U.S. Pat. Nos.5,591,669; 5,589,369; 5,545,807; and U.S Patent Publication Nos.20020199213 and 20030092125, which describe methods for biasing theimmune response of an animal to the desired epitope. Human antibodiesmay also be generated by in vitro activated B cells (see U.S. Pat. Nos.5,567,610 and 5,229,275).

Human antibodies can also be generated through the in vitro screening ofantibody display libraries. See Hoogenboom et al. (1991), J. Mol. Biol.227: 381; and Marks et al. (1991), J. Mol. Biol. 222: 581. Variousantibody-containing phage display libraries have been described and maybe readily prepared. Libraries may contain a diversity of human antibodysequences, such as human Fab, Fv, and scFv fragments that may bescreened against an appropriate target. Phage display libraries maycomprise peptides or proteins other than antibodies which may bescreened to identify selective binding agents of BoNT.

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments-usually Fv or Fabfragments-in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered either into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

Methods for display of peptides on the surface of yeast and microbialcells have also been used to identify antigen specific antibodies. See,for example, U.S. Pat. No. 6,699,658. Antibody libraries may be attachedto yeast proteins, such as agglutinin, effectively mimicking the cellsurface display of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingribosome mRNA display methods and microbial cell display methods.Selection of polypeptide using ribosome display is described in Hanes etal., (Proc. Nat.l Acad. Sc.i USA, 94:4937-4942, 1997) and U.S. Pat. Nos.5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is alsouseful for rapid large scale mutational analysis of antibodies. Theselective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

Human BoNT-neutralizing antibodies of the present disclosure are may beproduced in trioma cells. Genes encoding the antibodies are then clonedand expressed in other cells, particularly, nonhuman mammalian cells.

The general approach for producing human antibodies by trioma technologyhas been described by Ostberg et al. (1983) Hybridoma 2: 361-367,Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.4,634,666. The antibody-producing cell lines obtained by this method arecalled triomas because they are descended from three cells; two humanand one mouse. Triomas have been found to produce antibody more stablythan ordinary hybridomas made from human cells.

Other approaches to antibody production include in vitro immunization ofhuman blood. In this approach, human blood lymphocytes capable ofproducing human antibodies are produced. Human peripheral blood iscollected from the patient and is treated to recover mononuclear cells.The suppressor T-cells then are removed and remaining cells aresuspended in a tissue culture medium to which is added the antigen andautologous serum and, preferably, a nonspecific lymphocyte activator.The cells then are incubated for a period of time so that they producethe specific antibody desired. The cells then can be fused to humanmyeloma cells to immortalize the cell line, thereby to permit continuousproduction of antibody (see U.S. Pat. No. 4,716,111).

In another approach, mouse-human hybridomas which produce humanBoNT-neutralizing antibodies are prepared (see, e.g., U.S. Pat. No.5,506,132). Other approaches include immunization of murines transformedto express human immunoglobulin genes, and phage display screening(Vaughan et al. supra.).

VI. Other Antibody Forms.

Sequence provided herein can be used to generate other antibody forms,including but not limited to nanobodies, UniBodies, and/or affibodies.

VHH and/or Nanobodies.

The Camelidae heavy chain antibodies are found as homodimers of a singleheavy chain, dimerized via their constant regions. The variable domainsof these camelidae heavy chain antibodies are referred to as V_(HH)domains or V_(HH), and can be either used per se as nanobodies and/or asa starting point for obtaining nanobodies. Isolated V_(HH) retain theability to bind antigen with high specificity (see, e.g.,Hamers-Casterman et al. (1993) Nature 363: 446-448). V_(HH) domains, ornucleotide sequences encoding them, can be derived from antibodiesraised in Camelidae species, for example in camel, dromedary, llama,alpaca and guanaco. Other species besides Camelidae (e.g, shark,pufferfish) can produce functional antigen-binding heavy chainantibodies, from which (nucleotide sequences encoding) such naturallyoccurring V_(HH) can be obtained, e.g. using the methods described inU.S. Patent Publication US 2006/0211088.

Human proteins may be used in therapy primarily because they are not aslikely to provoke an immune response when administered to a patient.Comparisons of camelid V_(HH) with the V_(H) domains of human antibodiesreveals several key differences in the framework regions of the camelidV_(HH) domain corresponding to the V_(H)/V_(L) interface of the humanV_(H) domains. Mutation of these human residues to V_(HH) resemblingresidues has been performed to produce “camelized” human V_(H) domainsthat retain antigen binding activity, yet have improved expression andsolubility.

Libraries of single V_(H) domains have also been derived for examplefrom V_(H) genes amplified from genomic DNA or from mRNA from thespleens of immunized mice and expressed in E. coli (Ward et al. (1989)Nature 341: 544-546) and similar approaches can be performed using theV_(H) domains and/or the V_(L) domains described herein. The isolatedsingle V_(H) domains are called “dAbs” or domain antibodies. A “dAb” isan antibody single variable domain (V_(H) or V_(L)) polypeptide thatspecifically binds antigen. A “dAbV binds antigen independently of otherV domains; however, as the term is used herein, a “dAbU can be presentin a homo- or heteromultimer with other V_(H) or V_(L) domains where theother domains are not required for antigen binding by the dAb, i.e.,where the dAb binds antigen independently of the additional V_(H) orV_(L) domains.

As described in U.S. Patent Publication No. 2006/0211088 methods areknown for the cloning and direct screening of immunoglobulin sequences(including but not limited to multivalent polypeptides comprising: twoor more variable domains—or antigen binding domains—and in particularV_(H) domains or V_(HH) domains; fragments of V_(L), V_(H) or V_(HH)domains, such as CDR regions, for example CDR3 regions; antigen-bindingfragments of conventional 4-chain antibodies such as Fab fragments andscFv's, heavy chain antibodies and domain antibodies; and in particularof VH sequences, and more in particular of V_(HH) sequences) that can beused as part of and/or to construct such nanobodies.

Methods and procedures for the production of V_(HH)/nanobodies can alsobe found for example in WO 94/04678, WO 96/34103, WO 97/49805, WO97/49805 WO 94/25591, WO 00/43507 WO 01/90190, WO 03/025020, WO04/062551, WO 04/041863, WO 04/041865, WO 04/041862, WO 04/041867,PCT/BE2004/000159, Hamers-Casterman et al. (1993) Nature 363: 446;Riechmann and Muyldermans (1999) J. Immunological Meth., 231: 25-38; Vuet al. (1997) Molecular Immunology, 34(16-17): 1121-1131; Nguyen et al.(2000) EMBO J., 19(5): 921-930; Arbabi Ghahroudi et al. (19997) FEBSLetters 414: 521-526; van der Linden et al. (2000) J. ImmunologicalMeth., 240: 185-195; Muyldermans (2001) Rev. Molecular Biotechnology 74:277-302; Nguyen el al. (2001) Adv. Immunol. 79:261, and the like, whichare all incorporated herein by reference.

UniBodies.

UniBodies are generated by an antibody technology that produces astable, smaller antibody format with an anticipated longer therapeuticwindow than certain small antibody formats. UniBodies may be producedfrom IgG4 antibodies by eliminating the hinge region of the antibody.Unlike the full size IgG4 antibody, the half molecule fragment is verystable and is termed a UniBody. Halving the IgG4 molecule left only onearea on the UniBody that can bind to a target. Methods of producingUniBodies are described in detail in PCT Publication W02007/059782,which is incorporated herein by reference in its entirety (see, also,Kolfschoten et al. (2007) Science 317: 1554-1557).

Affibodies.

Affibody molecules are class of affinity proteins based on a 58-aminoacid residue protein domain, derived from one of the IgG-binding domainsof staphylococcal protein A. This three helix bundle domain has beenused as a scaffold for the construction of combinatorial phagemidlibraries, from which affibody variants that target the desiredmolecules can be selected using phage display technology (see, e.g, Nordet al. (1997) Nat. Biotechnol. 15: 772-777; Ronmark et al. (2002) Eur.J. Biochem., 269: 2647-2655.). Details of affibodies and methods ofproduction are known to those of skill (see, e.g., U.S. Pat. No.5,831,012 which is incorporated herein by reference in its entirety).

VI. Assaying for Cross-Reactivity at a Neutralizing Epitope.

The antibodies of the present disclosure encompass those thatspecifically bind to one or more epitopes recognized by antibodiesdescribed herein (e.g., 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5, 3E6,3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12, B1.1,B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18, 2B18.1,4B19, 4B19.1, 1B22, 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23, 2B24,2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C, 4B17.1D,4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2, B11.A5,B11.F7, B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1,5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2, etc.). In otherwords, antibodies are cross-reactive with one of more of theseantibodies. Means of assaying for cross-reactivity are well known tothose of skill in the art (see, e.g., Dowbenko et al. (1988) J. Virol.62: 4703-4711).

This can be ascertained by providing one or more isolated target BoNTpolypeptide(s) (e.g. BoNT/B1 and/or BoNT/B2, or recombinant domains ofsaid toxin, such as HO attached to a solid support and assaying theability of a test antibody to compete with, an antibody described hereinfor binding to the target BoNT peptide. Thus, immunoassays in acompetitive binding format are preferably used for cross-reactivitydeterminations. For example, a BoNT/E and/or BoNT/B polypeptide may beimmobilized to a solid support. Antibodies to be tested (e.g. generatedby selection from a phage-display library) added to the assay competewith 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 3E6.2, 4E11,4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1,B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18, 2B18.1, 4B19, 4B19.1, 1B22,1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23, 2B24, 2B25, 2B25.1, 2B26,2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C, 4B17.1D, 4B17.1F, 4B17.1G,3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2, B11.A5, B11.F7, B11.H12,B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1,ING1.5B1, ING1.2B10, and ING1.3C2, etc antibodies binding to theimmobilized BoNT polypeptide(s). The ability of test antibodies tocompete with the binding of the 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5,3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, A12, 6A12,B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18,2B18.1, 4B19, 4B19.1, 1B22, 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23,2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C,4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2,B11.A5, B11.F7, B11.H12, B11.E9, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1,4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, and ING1.3C2, etcantibodies to the immobilized protein(s) are compared. The percentcross-reactivity above proteins is then calculated, using standardcalculations.

If the test antibody competes with one or more of the 2A10, 3E1, 3E2,3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17,4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12,B12.1, B12.2, 1B18, 2B18.1, 4B19, 4B19.1, 1B22, 1B10, 1B10.1, 2B18.2,2B18.3, 1B22.4, 2B23, 2B24, 2B25, 2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30,4B17.1, 4B17.1C, 4B17.1D, 4B17.1F, 4B17.1G, 3E6.2, 4E17.4, 4E17.6,4B19.1, B6.C12, B6.D2, B11.A5, B11.F7, B11.H12, B11.E9, 4B1, 4B3, 4B5,4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, ING1.1C1, ING1.5B1, ING1.2B10, andING1.3C2, etc antibodies and has a binding affinity comparable to orgreater than about 1×10⁻⁸ M with the same target then the test antibodyis expected to be a BoNT-neutralizing antibody.

Cross-reactivity may performed by using surface plasmon resonance in aBIAcore. In a BIAcore flow cell, the BoNT polypeptide(s) (e.g., BoNT/Band/or BoNT/E) are coupled to a sensor chip (e.g. CM5) as described incopending application No. 60/942,173, disclosure of which isincorporated herein by reference. With a flow rate of 5 μl/min, atitration of 100 nM to 1 μM antibody is injected over the flow cellsurface for about 5 minutes to determine an antibody concentration thatresults in near saturation of the surface. Epitope mapping orcross-reactivity is then evaluated using pairs of antibodies atconcentrations resulting in near saturation and at least 100 RU ofantibody bound. The amount of antibody bound is determined for eachmember of a pair, and then the two antibodies are mixed together to givea final concentration equal to the concentration used for measurementsof the individual antibodies. Antibodies recognizing different epitopesshow an essentially additive increase in the RU bound when injectedtogether, while antibodies recognizing identical epitopes show only aminimal increase in RU. Antibodies may be said to be cross-reactive if,when “injected” together they show an essentially additive increase(preferably an increase by at least a factor of about 1.4, morepreferably an increase by at least a factor of about 1.6, and mostpreferably an increase by at least a factor of about 1.8 or 2.

Cross-reactivity may also be determined by incubating a yeast displayedscFv with a BoNT domain polypeptide followed by incubation with anepitope-tagged scFv. Bound scFv is detected with an antibody recognizingthe epitope tag and the level of BoNT domain display quantitated byincubation with anti-SV5 (see example 1).

Cross-reactivity at the desired epitopes can ascertained by a number ofother standard techniques (see, e.g., Geysen et al (1987) J. Immunol.Meth. 102, 259-274). This technique involves the synthesis of largenumbers of overlapping BoNT peptides. The synthesized peptides are thenscreened against one or more of the prototypical antibodies (e.g., 1B10, 3E6.2, etc.) and the characteristic epitopes specifically bound bythese antibodies can be identified by binding specificity and affinity.The epitopes thus identified can be conveniently used for competitiveassays as described herein to identify cross-reacting antibodies.

The peptides for epitope mapping can be conveniently prepared using“Multipin” peptide synthesis techniques (see, e.g., Geysen et al (1987)Science, 235: 1184-1190). Using the known sequence of one or more BoNTsubtypes (see, e.g., Atassi et al. (1996) J. Prot. Chem., 7: 691-700 andreferences cited therein), overlapping BoNT polypeptide sequences can besynthesized individually in a sequential manner on plastic pins in anarray of one or more 96-well microtest plate(s).

The procedure for epitope mapping using this multipin peptide system isdescribed in U.S. Pat. No. 5,739,306. Briefly, the pins are firsttreated with a pre-coat buffer containing 2% bovine serum albumin and0.1% Tween 20 in PBS for 1 hour at room temperature. Then the pins arethen inserted into the individual wells of 96-well microtest platecontaining the antibodies in the pre-coat buffer, e.g. at 2 μg/ml. Theincubation is preferably for about 1 hour at room temperature. The pinsare washed in PBST (e.g., 3 rinses for every 10 minutes), and thenincubated in the wells of a 96-well microtest plate containing 100 mu 1of HRP-conjugated goat anti-mouse IgG (Fc) (Jackson ImmunoResearchLaboratories) at a 1:4,000 dilution for 1 hour at room temperature.After the pins are washed as before, the pins are put into wellscontaining peroxidase substrate solution of diammonium 2,2′-azino-bis[3-ethylbenzthiazoline-b-sulfonate] (ABTS) and H₂O₂ (Kirkegaard & PerryLaboratories Inc., Gaithersburg, Md.) for 30 minutes at room temperaturefor color reaction. The plate is read at 405 nm by a plate reader (e.g.,BioTek ELISA plate reader) against a background absorption wavelength of492 nm. Wells showing color development indicate reactivity of the BoNTpeptides in such wells with the test antibodies.

VII. Assaying for Neutralizing Activity of Anti-BoNT Antibodies

Preferred antibodies of the present disclosure act, individually or incombination, to neutralize (reduce or eliminate) the toxicity ofbotulinum neurotoxin type. Neutralization can be evaluated in vivo or invitro. In vivo neutralization measurements simply involve measuringchanges in the lethality (e.g., LD₅₀ or other standard metric) due to aBoNT neurotoxin administration due to the presence of one or moreantibodies being tested for neutralizing activity. The neurotoxin can bedirectly administered to the test organism (e.g. mouse) or the organismcan harbor a botulism infection (e.g., be infected with Clostridiumbotulinum). The antibody can be administered before, during, or afterthe injection of BoNT neurotoxin or infection of the test animal. Adecrease in the rate of progression, or mortality rate indicates thatthe antibody(s) have neutralizing activity.

One suitable in vitro assay for neutralizing activity uses ahemidiaphragm preparation (Deshpande et al. (1995) Toxicon, 33:551-557). Briefly, left and right phrenic nerve hemidiaphragmpreparations are suspended in physiological solution and maintained at aconstant temperature (e.g. 36° C.). The phrenic nerves are stimulatedsupramaximally (e.g. at 0.05 Hz with square waves of 0.2 ms duration).Isometric twitch tension is measured with a force displacementtransducer (e.g., GrassModel FT03) connected to a chart recorder.

Purified antibodies are incubated with purified BoNT (e.g. BoNT/A1,BoNT/A2, BoNT/B1, etc.) for 30 min at room temperature and then added tothe tissue bath, resulting in a final antibody concentration of about2.0×10⁻⁸ M and a final BoNT concentration of about 2.0×10⁻¹¹ M. For eachantibody studied, time to 50% twitch tension reduction is determined(e.g., three times for BoNT alone and three times for antibody plusBoNT). Differences between times to a given (arbitrary) percentage (e.g.50%) twitch reduction are determined by standard statistical analyses(e.g. two-tailed t test) at standard levels of significance (e.g., a Pvalue of <0.05 considered significant).

VIII. Diagnostic Assays.

As explained above, the anti-BoNT antibodies of the present disclosurecan be used for the in vivo or in vitro detection of BoNT toxin (e.g.BoNT/E toxin) and thus, are useful in the diagnosis (e.g. confirmatorydiagnosis) of botulism. The detection and/or quantification of BoNT in abiological sample obtained from an organism is indicative of aClostridium botulinum infection of that organism.

The BoNT antigen can be quantified in a biological sample derived from apatient such as a cell, or a tissue sample derived from a patient. Asused herein, a biological sample is a sample of biological tissue orfluid that contains a BoNT concentration that may be correlated with andindicative of a Clostridium botulinum infection. Preferred biologicalsamples include blood, urine, saliva, and tissue biopsies.

Although the sample is typically taken from a human patient, the assayscan be used to detect BoNT antigen in samples from mammals in general,such as dogs, cats, sheep, cattle and pigs, and most particularlyprimates such as humans, chimpanzees, gorillas, macaques, and baboons,and rodents such as mice, rats, and guinea pigs.

Tissue or fluid samples are isolated from a patient according tostandard methods well known to those of skill in the art, most typicallyby biopsy or venipuncture. The sample is optionally pretreated asnecessary by dilution in an appropriate buffer solution or concentrated,if desired. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, at physiological pH can be used.

A) Immunological Binding Assays

The BoNT polypeptide (e.g., BoNT/E, BoNT/B, etc.) can be detected in animmunoassay utilizing one or more of the anti-BoNT antibodies of thepresent disclosure as a capture agent that specifically binds to theBoNT polypeptide.

As used herein, an immunoassay is an assay that utilizes an antibody(e.g. a anti-BoNT/E antibody) to specifically bind an analyte (e.g.,BoNT/E). The immunoassay is characterized by the binding of one or moreanti-BoNT antibodies to a target (e.g. one or more BoNT/E subtypes) asopposed to other physical or chemical properties to isolate, target, andquantify the BoNT analyte.

The BoNT marker can be detected and quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168, and the like). For areview of the general immunoassays, see also Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc.New York (1993); Basic and Clinical Immunology 7th Edition, Stites &Terr, eds. (1991)).

The immunoassays of the present disclosure can be performed in any of anumber of configurations (see, e.g., those reviewed in Maggio (ed.)(1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985)“Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers B.V.,Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay: APractical Guide Academic Press, Orlando, Fla.; Price and Newman (eds.)(1991) Principles and Practice of Immunoassays Stockton Press, NY; andNgo (ed.) (1988) Non isotopic Immunoassays Plenum Press, NY).

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the capture agent and the analyte(e.g., an anti-BoNT/E antibody/BoNT/E complex). The labeling agent canitself be one of the moieties comprising the antibody/analyte complex.Thus, for example, the labeling agent can be a labeled BoNT/Epolypeptide or a labeled anti-BoNT/E antibody. Alternatively, thelabeling agent is optionally a third moiety, such as another antibody,that specifically binds to the BoNT antibody, the BoNT peptide(s), theantibody/polypeptide complex, or to a modified capture group (e.g.,biotin) which is covalently linked to BoNT polypeptide or to theanti-BoNT antibody.

The labeling agent encompasses an antibody that specifically binds tothe anti-BoNT antibody. Such agents are well known to those of skill inthe art, and most typically comprise labeled antibodies thatspecifically bind antibodies of the particular animal species from whichthe anti-BoNT antibody is derived (e.g., an anti-species antibody).Thus, for example, where the capture agent is a human derived BoNT/Eantibody, the label agent may be a mouse anti-human IgG, i.e., anantibody specific to the constant region of the human antibody.

Other proteins capable of specifically binding immunoglobulin constantregions, such as streptococcal protein A or protein G are also used asthe labeling agent. These proteins are normal constituents of the cellwalls of streptococcal bacteria. They exhibit a strong non immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies (see generally Kronval, et al., (1973) J. Immunol.,111:1401-1406, and Akerstrom, et al., (1985) J. Immunol., 135:2589-2542,and the like).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays are carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 5° C. to 45° C.

1) Non Competitive Assay Formats.

Immunoassays for detecting BoNT neurotoxins (e.g. BoNT serotypes and/orsubtypes) may be either competitive or noncompetitive. Noncompetitiveimmunoassays are assays in which the amount of captured analyte (in thiscase, BoNT polypeptide) is directly measured. In one preferred“sandwich” assay, for example, the capture agent (e.g., an anti-BoNTantibody) is bound directly or indirectly to a solid substrate where itis immobilized. These immobilized anti-BoNT antibodies capture BoNTpolypeptide(s) present in a test sample (e.g., a blood sample). The BoNTpolypeptide(s) thus immobilized are then bound by a labeling agent,e.g., an anti-BoNT/E antibody bearing a label. Alternatively, the secondantibody may lack a label, but it may, in turn, be bound by a labeledthird antibody specific to antibodies of the species from which thesecond antibody is derived. Free labeled antibody is washed away and theremaining bound labeled antibody is detected (e.g., using a gammadetector where the label is radioactive).

2) Competitive Assay Formats.

In competitive assays, the amount of analyte (e.g., BoNT/E) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte displaced (or competed away) from a capture agent(e.g., anti-BoNT/E antibody) by the analyte present in the sample. Forexample, in one competitive assay, a known amount of BoNT/E is added toa test sample with an unquantified amount of BoNT/E, and the sample iscontacted with a capture agent, e.g., an anti-BoNT/E antibody thatspecifically binds BoNT/E. The amount of added BoNT/E that binds to theanti-BoNT/E-neutralizing antibody is inversely proportional to theconcentration of BoNT/E present in the test sample.

The anti-BoNT/E antibody can be immobilized on a solid substrate. Theamount of BoNT/E bound to the anti-BoNT/E antibody is determined eitherby measuring the amount of BoNT/E present in a BoNT/E-anti-BoNT/Eantibody complex, or alternatively by measuring the amount of remaininguncomplexed BoNT/E.

B) Reduction of Non Specific Binding.

One of skill will appreciate that it is often desirable to reduce nonspecific binding in immunoassays and during analyte purification. Wherethe assay involves, for example BoNT/E polypeptide(s),BoNT/E-neutralizing antibody, or other capture agent(s) immobilized on asolid substrate, it is desirable to minimize the amount of non specificbinding to the substrate. Means of reducing such non specific bindingare well known to those of skill in the art. Typically, this involvescoating the substrate with a proteinaceous composition. In particular,protein compositions such as bovine serum albumin (BSA), nonfat powderedmilk, and gelatin are widely used.

C) Substrates.

As mentioned above, depending upon the assay, various components,including the BoNT polypeptide(s), anti-BoNT antibodies, etc., areoptionally bound to a solid surface. Many methods for immobilizingbiomolecules to a variety of solid surfaces are known in the art. Forinstance, the solid surface may be a membrane (e.g., nitrocellulose), amicrotiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube(glass or plastic), a dipstick (e.g., glass, PVC, polypropylene,polystyrene, latex, and the like), a microcentrifuge tube, or a glass,silica, plastic, metallic or polymer bead. The desired component may becovalently bound, or noncovalently attached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

In preparing the surface, a plurality of different materials may beemployed, e.g., as laminates, to obtain various properties. For example,protein coatings, such as gelatin can be used to avoid non specificbinding, simplify covalent conjugation, enhance signal detection or thelike.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas, (1970) J. Biol. Chem.245 3059.

In addition to covalent bonding, various methods for noncovalentlybinding an assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface. Typically, thesurface is blocked with a second compound to prevent nonspecific bindingof labeled assay components. Alternatively, the surface is designed suchthat it nonspecifically binds one component but does not significantlybind another. For example, a surface bearing a lectin such asconcanavalin A will bind a carbohydrate containing compound but not alabeled protein that lacks glycosylation. Various solid surfaces for usein noncovalent attachment of assay components are reviewed in U.S. Pat.Nos. 4,447,576 and 4,254,082.

D) Other Assay Formats

BoNT polypeptides or anti-BoNT antibodies (e.g. BoNT/E neutralizingantibodies) can also be detected and quantified by any of a number ofother means well known to those of skill in the art. These includeanalytic biochemical methods such as spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, and various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.

Western blot analysis and related methods can also be used to detect andquantify the presence of BoNT polypeptides in a sample. The techniquegenerally comprises separating sample products by gel electrophoresis onthe basis of molecular weight, transferring the separated products to asuitable solid support, (such as a nitrocellulose filter, a nylonfilter, or derivatized nylon filter), and incubating the sample with theantibodies that specifically bind either the BoNT polypeptide. Theantibodies specifically bind to the biological agent of interest on thesolid support. These antibodies are directly labeled or alternativelyare subsequently detected using labeled antibodies (e.g., labeled sheepanti-human antibodies where the antibody to a marker gene is a humanantibody) which specifically bind to the antibody which binds the BoNTpolypeptide.

Other assay formats include liposome immunoassays (LIAs), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,(1986) Amer. Clin. Prod. Rev. 5:34-41).

E) Labeling of Anti-BoNT (e.g., Anti-BoNT/E) Antibodies.

Anti-BoNT antibodies can be labeled by any of a number of methods knownto those of skill in the art. Thus, for example, the labeling agent canbe, e.g., a monoclonal antibody, a polyclonal antibody, a protein orcomplex such as those described herein, or a polymer such as an affinitymatrix, carbohydrate or lipid. Detection proceeds by any known method,including immunoblotting, western analysis, gel-mobility shift assays,tracking of radioactive or bioluminescent markers, nuclear magneticresonance, electron paramagnetic resonance, stopped-flow spectroscopy,column chromatography, capillary electrophoresis, or other methods whichtrack a molecule based upon an alteration in size and/or charge. Thedetectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, any label useful in suchmethods can be applied in the various embodiments of the presentdisclosure. Thus, a label is any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Useful labels in the present disclosureinclude magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g.,fluorescein isothiocyanate, Texas red, rhodamine, ALEXA FLUOR dyes andthe like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes(e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase andothers, commonly used as detectable enzymes, either as marker geneproducts or in an ELISA), and colorimetric labels such as colloidal goldor colored glass or plastic (e.g. polystyrene, polypropylene, latex,etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, ease of conjugation of thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, e.g., by microscopy,visual inspection, via photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple colorimetric labels may be detectedsimply by observing the color associated with the label. Thus, invarious dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence ofBoNT peptides. In this case, antigen-coated particles are agglutinatedby samples comprising the target antibodies. In this format, none of thecomponents need be labeled and the presence of the target antibody isdetected by simple visual inspection.

IX. Compositions.

The BoNT-neutralizing antibodies of this disclosure are useful inmitigating the progression of botulism produced, e.g., by endogenousdisease processes or by chemical/biological warfare agents. Typicallycompositions comprising one, two, or more different antibodies can beprovided as a pharmaceutical composition and administered to a mammal(e.g., to a human) in need thereof.

As disclosed herein, particularly efficient neutralization of a botulismneurotoxin (BoNT) subtype is achieved by the use of neutralizingantibodies that bind two or more subtypes of the particular BoNTserotype with high affinity. This can be accomplished by using two ormore different antibodies directed against each of the subtypes and/orneutralizing antibodies that bind two or more BoNT subtypes (e.g.,BoNT/B1, BoNT/B2, BoNT/B3, etc.) with high affinity.

Different neutralizing antibodies when combined, exhibit a potency thatis increased dramatically. This increase makes it possible to generate abotulinum antibody composition of the required potency for therapeuticuse. It was also surprising that as one begins combining two and threemonoclonal antibodies, the particular BoNT epitope that is recognizedbecomes less important. Compositions comprising at least two, at leastthree, or more high affinity antibodies that bind overlapping ornon-overlapping epitopes on the BoNT are contemplated herein.

Compositions contemplated herein may contain two, three, or moredifferent antibodies selected from the following: 2A10, 3E1, 3E2, 3E3,3E4, 3E4.1, 3E5, 3E6, 3E6.1, 3E6.2, 4E11, 4E13, 4E16, 4E16.1, 4E17,4E17.1, A12, 6A12, B1.1, B6, B6.1, B6.C12, B6.D2, B8, B8.1, B11, B11C3,B11E8, B11.A5, B11.F7, B11.H12, B12, B12.1, B12.2, 1B18, 2B18.1, 4B19,4B19.1, 1B22, 1B10, 1B10.1, 2B18.2, 2B18.3, 1B22.4, 2B23, 2B24, 2B25,2B25.1, 2B26, 2B27, 2B28, 2B29, 2B30, 4B17.1, 4B17.1C, 4B17.1D, 4B17.1F,4B17.1G, 3E6.2, 4E17.4, 4E17.6, 4B19.1, B6.C12, B6.D2, B11.A5, B11.F7,B.H12, 4B1, 4B3, 4B5, 4B6, 4B7, 1B14, 4A1, 4A1.1, 5A20.4, 1C1, 5B1,2B10, B11.E9, and 3C2. The composition may optionally further includeantibodies comprising one or more CDRs from these antibodies, and/or oneor more antibodies comprising mutants or derivatives of theseantibodies. Exemplary compositions of the present disclosure contains atleast 2, at least 3, or at least 4 of the antibodies B10.1, 2B18.1B11E8, B6.1, B12.1 or of the anitbodies 3E6.2, 4E17.1, 4E16.1, 3E2,which antibodies can be provided in combination with a pharmaceuticalcarrier.

An exemplary composition for binding BoNT/B includes 1B10.1, 2B18.1, andB11E8. 2B18.3 may be optionally included in addition to this exemplarycomposition. 2B18.1 may also be optionally substituted with 2B18.3 andvice versa. B11E8 may also be substituted with B12.1, 4B19.1, 2B23, or1B22. This composition may further include B6.1 and/or B8.1. Suchcompositions can find use in neutralizing BoNT/B.

An exemplary composition for binding BoNT/E includes 4E17.1 and two ormore antibodies from the following: 4E16.1, 3E2, 3E6.2, and 3E6.1. 3E6.1may optionally be substituted with 3E6.2 and vice versa. Suchcompositions can find use in neutralizing BoNT/E.

The subject composition encompasses compositions that specificallyneutralize one serotype, such as serotype BoNT/A, BoNT/B, or BoNT/E. Thecomposition may also contain any first combination of antibodiesdescribed above that specifically neturalizes one serotype together witha second combination of antibodies that specifically neutralizes adifferent serotype. The subject composition may contain multiplecombinations such that that composition may neturalize two, three, ormore serotypes (e.g. BoNT/A, BoNT/B, and/or BoNT/E).

An exemplary composition that neutralizes multiple serotypes may includeany of the combinations described above or one or more of the antibodiesdisclosed in Tables 1-5, together with one or more of the antibodiesthat bind to BoNT/A. Antibodies that bind to BoNT/A that may be includedin the subject composition encompass those that have one or more CDRs orfull-length V_(H) or V_(L) from clone 3D12, RAZ1, CR2, or 2G11, listedin the table below.

TABLE 7 Amino acid sequences for V_(H) or V_(L) CDRs of antibodies of BoNT/Aclone V_(H) CDR1 V_(H) CDR2 V_(H) CDR3 3D12 DYDMH VMWFDGTEKYSAESVKGEPDWLLWGDRGALDV (SEQ (SEQ ID NO: 873) (SEQ ID NO: 874) ID NO: 875) RAZ1DYDMH  VMWFDGTEKYSAESVKG EPDWLLWGDRGALDV (SEQ (SEQ ID NO: 876)(SEQ ID NO: 877) ID NO: 878) CR2 YDYMY  TISDGGSYTYYSDSVEGYRYDDAMDY (SEQ ID (SEQ ID NO: 879) (SEQ ID NO: 880) NO: 881) 2G11 NYAMT SISVGGSDTYYADSVKG VRTKYCSSLSCFAGFDS (SEQ ID NO: 882) (SEQ ID NO: 883)(SEQ ID NO: 884) V_(L) CDR1 V_(L) CDR2 V_(L) CDR3 3D12 RASQSISSWLAEASSLES QHYNTYPYT (SEQ ID (SEQ ID NO: 885) (SEQ ID NO: 886) NO: 887)RAZ1 WASQSISSRLA EATSLGS QHYDTYPYT (SEQ ID (SEQ ID NO: 888)(SEQ ID NO: 889) NO: 890) CR2 RASESVDSYGHSFMQ RASNLEP QQGNEVPFT (SEQ ID(SEQ ID NO: 891) (SEQ ID NO: 892) NO: 893) 2G11 RASQSISSYLH DASSSQS QQSYSTRALT (SEQ ID (SEQ ID NO: 894) (SEQ ID NO: 895) NO: 896)

For example, a combination of antibodies that neutralize BoNT/A that maybe in the subject compositon may include RAZ1, CR2, and 2G11. Such acombination that neutralizes BoNT/A may be included in a compositioncontaining the various exemplary combinations described above toneutralize BoNT/B or BoNT/E. As an example of a composition thatneutralizes both BoNT/A and BoNT/B, the composition may include RAZ1,CR2, and 2G11 together with 1B10.1, 2B18.1, and B11E8, with any optionaladdition or substitutions mentioned above. One optional substitution ofany of RAZ1, CR2, and 2G11 may be 4E17.1. 4E17.1 may also be added to acomposition used to neturalize BoNT/A. Where a composition neutralizesall BoNT/A, BoNT/B, and BoNT/E, the composition may contain RAZ1, CR2,2G11, 1B10.1, 2B18.1, B11E8, 4E17.1, together with two or more of4E16.1, 3E2, 3E6.2 and/or 3E6.1. Similarly, any composition describedherein may modified to include additional antibodies or to have anantibody substituted with another (e.g. derivative thereof).

Where combinations of antibodies are disclosed herein, such combinationscan be provided in a single formulation or can be provided as separateformulations in a kit, where the separate formulations may contain asingle antibody or two antibodies. Such separate formulations of a kitmay be combined prior to administration or administered by separateinjection.

The BoNT-neutralizing antibodies provided by the present disclosure areuseful for parenteral, topical, oral, or local administration, such asby aerosol or transdermally, for prophylactic and/or therapeutictreatment. The pharmaceutical compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and lozenges.The antibodies comprising the pharmaceutical compositions of the presentdisclosure, when administered orally, are preferably protected fromdigestion. This is typically accomplished either by complexing theantibodies with a composition to render them resistant to acidic andenzymatic hydrolysis or by packaging the antibodies in an appropriatelyresistant carrier such as a liposome. Means of protecting proteins fromdigestion are well known in the art.

The pharmaceutical compositions of the present disclosure areparticularly useful for parenteral administration, such as intravenousadministration or administration into a body cavity or lumen of anorgan. The compositions for administration will commonly comprise asolution of one or more BoNT-neutralizing antibody dissolved in apharmaceutically acceptable carrier, which may be an aqueous carrier. Avariety of aqueous carriers can be used, e.g., buffered saline and thelike.

Non-aqueous pharmaceutically acceptable carriers (excipients) are knownto those of skill in the art. Such excipients, can comprise anysubstance that is biocompatible and liquid or soft enough at thesubject's body temperature to release the active agent(s) (e.g.,antibodies) somatotropin into the subject's bloodstream at a desiredrate. Non-aqueous carriers are usually hydrophobic and commonly organic,e. g., an oil or fat of vegetable, animal, mineral or synthetic originor derivation. The carrier may include at least one chemical moiety ofthe kind that typifies “fatty” compounds, e. g., fatty acids, alcohols,esters, etc., i. e., a hydrocarbon chain, an ester linkage, or both.“Fatty” acids in this context include, but are not limited to, acetic,propionic and butyric acids through straight- or branched-chain organicacids containing up to 30 or more carbon atoms. The non-aqueous carriermay be immiscible in water and/or soluble in the substances commonlyknown as fat solvents. The non-aqueous carrier can correspond to areaction product of a “fatty” compound or compounds with a hydroxycompound, e, g., a mono-hydric, di-hydric, trihydric or other polyhydricalcohol, e.g., glycerol, propanediol, lauryl alcohol, polyethyleneor-propylene glycol, etc. These compounds include, but are not limitedto, the fat-soluble vitamins, e.g., tocopherols and their esters, e. g.,acetates sometimes produced to stabilize tocopherols. Sometimes, foreconomic reasons, the carrier can comprise a natural, unmodifiedvegetable oil such as sesame oil, soybean oil, peanut oil, palm oil, oran unmodified fat. Alternatively the vegetable oil or fat may bemodified by hydrogenation or other chemical means which is compatiblewith the present disclosure. The appropriate use of hydrophobicsubstances prepared by synthetic means is also envisioned. Non-aqueousexcipient compositions can also comprise, in addition to a biocompatibleoil, an “antihydration agent” which term as used herein means asubstance that retards hydration of the active agent(s) and/or thebiocompatible oil or fat and thereby further decreases and/or stabilizesthe rate of release of the active agent(s) from that compositionfollowing administration to an animal. A great variety of non-toxicantihydration agents are known. By way of example there are “gelling”agents that, when dispersed, and in some cases heated to dissolve themin the oil, give the body of oil greater visco-elasticity (and thereforegreater structural stability) and thereby slow down penetration of theoil by body fluids.

Illustrative antihydration agents include various polyvalent metal saltsor complexes of organic acids, for instance fatty acids having fromabout 8 or 10 to about 20 or 22 carbon atoms, e. g. aluminum, zinc,magnesium or calcium salts of lauric acid, palmitic acid, stearic acidand the like. Such salts can be mono-, di- or tri-substituted, dependingon the valence of the metal and the degree of oxidation of the metal bythe acid. Of common usage are the aluminum salts of such fatty acids.Aluminum monostearate and distearate are frequently used anti-hydrationagents. Others that are useful include aluminum tristearate, calciummono- and distearate, magnesium mono- and distearate and thecorresponding palmitates, laurates and the like. The concentration ofsuch an antihydration agent, based on the weight of the oil plus thatagent, may be between about 1% and about 10% (most typically betweenabout 2% and about 5%), although other concentrations may be suitable insome cases.

The various solutions are sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like. Theconcentration of BoNT-neutralizing antibody in these formulations canvary widely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom about 1 mg up to about 200 mg per patient per day can be used.Methods for preparing parenterally administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa. (1980).

The compositions containing the BoNT-neutralizing antibodies of thepresent disclosure or a cocktail thereof are generally administered fortherapeutic treatments. Preferred pharmaceutical compositions areadministered in a dosage sufficient to neutralize (mitigate oreliminate) the BoNT toxin(s) (i.e., reduce or eliminate a symptom ofBoNT poisoning (botulism)). An amount adequate to accomplish this isdefined as a “therapeutically effective dose.” Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the antibodies of the present disclosure toeffectively treat the patient.

X. Kits For Diagnosis or Treatment.

Kits for the treatment of botulism or for the detection/confirmation ofa Clostridium botulinum infection are also provided. Kits will typicallycomprise one or more anti-BoNT antibodies (e.g., BoNT-neutralizingantibodies for pharmaceutical use). For diagnostic purposes, theantibody(s) can optionally be labeled. In addition the kits willtypically include instructional materials disclosing means of useBoNT-neutralizing antibodies in the treatment of symptoms of botulism.The kits may also include additional components to facilitate theparticular application for which the kit is designed. Thus, for example,where a kit contains one or more anti-BoNT antibodies for detection ofdiagnosis of BoNT subtype, the antibody can be labeled, and the kit canadditionally contain means of detecting the label (e.g. enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a sheep anti-humanantibodies, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

Kits provided for the treatment of botulism may contain one or more BoNTneutralizing antibodies. The antibodies can be provided separately ormixed together. Typically the antibodies will be provided in a sterilepharmacologically acceptable excipient. The antibodies can also beprovided pre-loaded into a delivery device (e.g., a disposable syringe).

The kits can optionally include instructional materials teaching the useof the antibodies, recommended dosages, contraindications, and the like.

EXAMPLES

The following examples are offered to illustrate, but not to limit anyembodiments provided by the present disclosure.

Example 1 Human Monoclonal Antibodies to Botulinum Neurotoxin Types Aand B from Immune Yeast Displayed Antibody Libraries

The following methods and materials were used in the present example.

Methods and Materials

Oligonucleotide primers Primary library construction HuVH2bBACK: (SEQ ID NO: 897) 5′-CAGGTCACCTTGAAGGAGTCTGG-3′ HuVH5bBACK: (SEQ ID NO: 898) 5′-GAGGTGCAGCTGGTGCAGTCTGG-3′ HuVH7aBACK: (SEQ ID NO: 899) 5′-CAGGTGCAGCTGGTGCAATCTGG-3′ HuVK2aBACK: (SEQ ID NO: 900) 5′-GATATTGTGATGACTCAGTCTCC-3′ HuVK2bBACK: (SEQ ID NO: 901) 5′-GATATTGTGATGACCCAGATCCC-3′Light chain shuffled library construction GAP5-HuRJH1-2BACK: (SEQ ID NO: 902) 5′-GTG GTG GTG GTT CTG CTA GCG GGG CCA TGG  CCACCC TGG TCA CCG TCT CCT CA -3′ GAP5-HuRJH3BACK:  (SEQ ID NO: 903)5′-GGT GGT GGT TCT GCT AGC GGG GCC ATG GC G ACA ATG  GTC ACC GTC TCT TCA-3′ GAP5-HuRJH4-5 BACK:  (SEQ ID NO: 904)5′-GGT GGT GGT TCT GCT AGC GGG GCC ATG GC A ACC CTG GTC ACC GTC TCC TCA-3  GAP5-HuRJH6 BACK:  (SEQ ID NO: 905)5′-GGT GGT GGT TCT GCT AGC GGG GCC ATG GC G ACC ACG  GTC ACC GTC TCC TCA-3  PYDFOR1:  (SEQ ID NO: 906) 5′-GTCGATTTTGTTACATCTACAC-3′V_(H) gene amplification from pYD2 pYD BACK1:  (SEQ ID NO: 907)5′-AGTAACGTTTGTCAGTAATTGC-3′

The underlined segment is the reverse complement of the JHFor primers

Other Primers

Other primers include the family of primers that used to create theVL-rep library (should anneal to the different Vk and Vl genes are addthe scFv linker, VHFR4, Nco1-Xho1 site. Also used is a family of primersto amplify the VH gene to clone into this vector, with 5′ overhang and3′ overhang for gap repair into VL-rep library.

Strains, Media Antibodies, and Toxins

Yeast strain Saccharomyces cerevisiae EBY100 (GAL1-AGA1TURA3 ura3-52trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1.41.6R canI) was maintained in YPDmedium (1% yeast extract, 2% peptone, 2% dextrose) (Current Protocols inMolecular Biology, John Wiley and Sons, Chapter 13.1.2). EBY100transformed with expression vector pYD2 (Razai et al., J Mol Biol. 2005,351:158-69) was selected on SD-CAA medium (0.7% yeast nitrogen base,0.1M Sodium phosphate, 0.5% casamino acids, 2% dextrose, 0.006%Leucine). ScFv yeast surface display was induced by transferring yeastcultures from SD-CAA to SG-CAA medium (identical to SD-CAA medium exceptthe glucose was replaced by galactose) and growing at 18° C. for 24 48hr as described previously (Feldhaus et al. Nat Biotechnol. 2003,21:163-70). Bacteria strain E. coli DH5α, (1(12, Δ(lac-pro), supE, thi,hsdD5/F′ traD36, proA+B+, lacIq, lacZΔM15) was used for cloning andpreparation of plasmid DNA. Pure BoNT/A1 (Hall hyper), BoNT/A2(FRI-H1A2), BoNT/B1, BoNT/B2, BoNT/B3, and BoNT/B4 were purified fromtheir respective strains or purchased from Metabiologics (Madison,Wis.). Complex BoNT/A3 was expressed and purified from strains A254 andBa207 (Hill et al. J Bacteriol. 2007, 189:818-32). Mouse anti-SV5 andanti-myc 9E10 antibody and BoNT/A antibodies 7C1 and 9D8 were purifiedfrom hybridoma supernatant using Protein G and directly labeled withALEXA FLUOR 488 dye or ALEXA FLUOR 647 dye using a kit provided by themanufacturer (MOLECULAR PROBES). Recombinant human BoNT/A antibodies3D12 and AR2 and BoNT/B antibodies A12 and 6A12 were purified fromChinese hamster ovary cells (CHO) supernatants (Nowakowski et al. ProcNatl Acad Sci USA. 2002, 99:11346-50; Razai et al., J Mol Biol. 2005,351:158-69). For flow cytometry (FACS), purified human or mouse IgGswere directly labeled with either ALEXA FLUOR 647 dye or ALEXA FLUOR 488dye using a kit provided by the manufacturer (MOLECULAR PROBES).

Immune scFv Yeast Antibody Library Generation

scFv yeast libraries were constructed from human volunteers immunizedwith pentavalent toxoid. Briefly approximately 25 ml of blood was drawn7 days after immunization and PBLs isolated using LYMPHOPREP tubes(Axis-Shield PoC AS, Norway). Blood was drawn after obtaining informedconsent and under a protocol reviewed and approved by the USAMRIID andUCSF IRBs. Total RNA was isolated from PBLs using an RNAgents® kit(Promega). cDNA was synthesized from total RNA by using AMV reversetranscriptase (Invitrogen) and HuIgGl-4-C1FOR, HuCK1FOR primers(Amersdorfer et al. Vaccine 2002, 20:1640-8; Marks et al. Eur J Immunol.1991, 21:985-91). V_(H) and V_(K) gene fragments were amplified by PCRfrom cDNA by using Pfu polymerase (Stratagene) and a mixture ofHuVH1-6BACK, HuVH2bBACK, HuVH5bBACK, HuVH7aBACK and HuJH1-5FOR mixprimers for the V_(H) gene and HuVK1-6BACK, HuVK2aBACK, HuVK2bBACK andHuJK1-5FOR primers or the V_(k) gene (Marks et al. 1991, supra). scFvlinker DNA template was prepared as previously described (Marks et al.1991, supra). PCR fragments were gel purified, isolated from the gelusing GENECLEAN® Turbo (Q.BIOgene). scFv gene repertoires wereconstructed by using PCR to splice together the V_(H) and V_(K) generepertoires with scFv linker DNA (Marks et al. 1991, supra). The scFvgenes were gel purified, isolated from the gel and reamplified usingHuVHBACK and HuJkFOR primer mixes which appended NcoI and NotI siterestriction sites (Marks et al. 1991, supra). scFv genes were digestedwith NcoI an NotI and ligated into NcoI-NotI digested pYD2 vector (Razaiet al. 2005, supra). scFv gene repertoires were amplified from theligation mixtures using primers GAPS and GAP3 (Razai et al. 2005, supra)to append homologous overlaps with pYD vector DNA. Appended scFv PCRproducts were ethanol precipitated, combined with NcoI-NotI digestedpYD2 and used to transform EBY100. Transformed yeast were cultured andsubcultured in SDCAA and library size calculated by serially dilutingand plating the transformed culture on SDCAA plates. Final library sizewas calculated as the product of the number of transformants and thepercentage of clones with full length scFv insert as determined by PCR(Razai et al., 2005, supra).

Selection and Characterization of Lead BoNT/B and BoNT/A scFv Antibodies

For antibody selection, forty times the library size was grown and scFvdisplay induced. For sorting, libraries were incubated with BoNT/A orBoNT/B in five times excess antigen, assuming each yeast expresses 1×10⁵scFv on the surface, at room temperature (RT) for 1 hr. For BoNT/Bsorting, the first two rounds were done by staining with 50-100 nMBoNT/B1 and the third and fourth round of staining were done by stainingwith 1-10 nM BoNT/B1. Libraries were washed once with ice cold FACSbuffer (PBS (1.9 mM NaH₂PO₄, 8.1 mM Na₂HPO₄, 154 mM NaCl₂, pH7.4), 0.5%BSA, 1 mM MgCl₂, 0.5 mM CaCl₂). BoNT/B binding was detected by stainingwith using 2.5 μg/ml of an equimolar mixture of mAbs A12 and 6A12labeled with ALEXA FLUOR 647 dye for 1 hr. at 4° C. Simultaneously, scFvdisplay level was quantitated by staining with 2.5 μg/ml anti-SV5 mAblabeled with ALEXA FLUOR 488 dye. Yeast libraries were washed asdescribed above, resuspended in 200 to 700 FACS buffer and were sortedon a FACSARIA cell sorter (Becton-Dickinson) with sort gates set tocollect all SV5 positive BoNT/B binding yeast. After the last round ofsorting, yeast were plated on SD-CAA plates and individual clones grownand induced. Individual clones were screened to identify BoNT/B bindingscFv and unique clones identified by PCR fingerprinting and DNAsequencing (Amersdorfer et al. 2002, supra). For each unique clone, theaffinity of the yeast displayed scFv for BoNT/B1, B2, B3, and B4 wasdetermined exactly as previously described (Razai et al. 2005, supra).

For one set of BoNT/A sortings, the first two rounds of sorting weredone by staining with 100 nM BoNT/A1 and 20 nM BoNT/A2 respectively.Libraries were washed once with ice cold FACS buffer (PBS (1.9 mMNaH₂PO₄, 8.1 mM Na₂HPO₄, 154 mM NaCl₂, pH7.4), 0.5% BSA, 1 mM MgCl₂, 0.5mM CaCl₂). BoNT/A binding was detected by staining with using 2.5 μg/mlof an equimolar mixture of mAbs 7C1 and 9D8 labeled with ALEXA FLUOR 647dye for 1 hr. at 4° C. Simultaneously, scFv display level wasquantitated by staining with 2.5 μg/ml anti-SV5 mAb labeled with ALEXAFLUOR 488 dye. Yeast libraries were washed as described above,resuspended in 200 to 700 μg FACS buffer and were sorted on a FACSARIAcell sorter (Becton-Dickinson) with sort gates set as in FIG. 8. For thelast two rounds of sorting, yeast libraries were incubated with either20 nM BoNT/A1 or 20 nM BoNT/A2 (see FIG. 8), washed, then stained with acombination of 2.5 μg/ml of AR1-ALEXA FLUOR 647 dye and 3D12-ALEXA FLUOR488 dye. Sort gates were set to capture only yeast binding BoNT/A whichwas bound by both 3D12 and AR1, thus ensuring that any resulting scFvwould bind BoNT/A epitopes that did not overlap with these two mAbs. Fora second set of BoNT/A sortings, the first three rounds of sorting weredone by staining with 100 nM BoNT/A1, the third and fourth round ofsorting were done by staining with 50 nM BoNT/A1, and the final round ofsorting was done by staining with 50 nM recombinant BoNT/A1 L_(C) orBoNT/A1 H_(N). For the first four rounds of sorting, BoNT/A binding wasdetected by staining with using 2.54 ml of an equimolar mixture of mAbs7C1 and 9D8 labeled with ALEXA FLUOR 647 dye for 1 hr at 4° C. For thefinal round of sorting, BoNT/A binding was detected by staining with2.54 ml of either mAbs 7C1 (for L_(C) incubation) or 9D8 (for H_(N)incubation) labeled with ALEXA FLUOR 647 dye for 1 hr. at 4° C.

After the last round of sorting, yeast were plated on SD-CAA plates andindividual clones grown and induced. Individual clones were screened toidentify BoNT/A binding scFv and unique clones identified by PCRfingerprinting and DNA sequencing (Amersdorfer et al. 2002, supra). Foreach unique clone, the affinity of the yeast displayed scFv for BoNT/A1and BoNT/A2 was determined exactly as previously described (Razai et al.2005, supra).

Construction and Expression of Yeast Displayed BoNT/B Domains andMapping of Antibody Binding

Primers HCFor, HCRev, HCFor, HNRev, and LCFor and LCRev were designed toPCR amplify the BoNT/B H_(C), H_(N), or L_(C) gene fragment respectivelyfrom the vectors, adding the restriction sites NcoI and NotI. Followingdigestion of both pYD2 and the resulting PCR amplification product withNcoI and NotI, the BoNT/B gene fragments were gel-purified and ligatedinto pYD2. Ligation mixtures were used to transform E. coli DH5α andcorrect clones identified by DNA sequencing. Plasmid DNA was used totransform LiAc-treated EBY100 cells. Yeast cultures were then grown andinduced, as described above. For mapping scFv binding to yeast displayedBoNT/B domains, scFv genes in pYD2 were excised by digestion with NcoIand NotI, ligated into pSYN1, and the ligation mixture used to transformE. coli TG1. scFv expression was induced and the periplasmic fractioncontaining the scFv prepared as previously described. Periplasmicfractions containing scFv were incubated for 1 hour at RT with yeastdisplaying BoNT/B domains. After washing with PBS, yeast were incubatedwith 1 ug/ml of mAb 9E10 which recognizes an epitope tag at the scFvC-terminus. After washing, 9E10 binding was detected using 1 ug/mlanti-mouse gammal specific antibody and the level of BoNT domain displayquantitated by incubation with 1 μg/ml of anti-SVS-ALEXA FLUOR 488 dye.For some antibodies, mapping was performed by staining with 1 μg/ml ofthe respective IgG conjugated to ALEXA FLUOR 647 dye.

Mapping the Overlap of BoNT Antibodies

Each yeast displayed scFv was grown and induced and incubated with 100nM BoNT/B for 1 hour at RT followed by washing with PBS. Afterresuspension, yeast were incubated with scFv containing periplasmicpreparations (see above), followed by washing and then incubation with 1μg/ml 9E10. After washing, 9E10 binding was detected using 1 ug/mlanti-mouse gammal specific antibody and the level of BoNT domain displayquantitated by incubation with 1 μg/ml of anti-SV5-ALEXA FLUOR 488 dye.For some antibodies, mapping was performed by staining with 1 μg/ml ofthe respective IgG conjugated to ALEXA FLUOR 647 dye.

Construction and Sorting of Chain Shuffled Yeast Antibody Libraries

To facilitate light chain shuffling, a light chain library was createdin the yeast display vector pYD2 by cloning in V_(L) gene repertoiresfrom donors six, nine, and ten. cDNA was synthesized from total RNAprepared from donor PBLs by using AMV reverse transcriptase (Invitrogen)and HuCK1FOR primers (Amersdorfer et al. 2002, supra; Marks et al. 1991,supra). V_(K) gene fragments were amplified by PCR from cDNA by usingPfu polymerase (Stratagene) and a mixture of HuVK1-6BACK, HuVK2aBACK,HuVK2bBACK and HuJK1-5FOR primers (Marks et al. 1991, supra). To furtherincrease light chain diversity, the light chain repertoire from a largenon-immune scFv phage antibody library was also utilized (Sheets et al.Proc Natl Acad Sci USA. 1998, 95:6157-62). Phage antibody libraryplasmid DNA was prepared and the VL genes PCR amplified using primersHuVK1-6BACK, HuVK2aBACK, HuVK2bBACK and HuJK1-5FOR for Vk genes andprimers V1 genes. PCR fragments were gel purified and isolated from thegel using GENECLEAN® Turbo (Q.BIOgene). V_(L) genes were reamplified byusing PCR primers to append the 3′ end of the V_(H) framework 4,containing a Xho1 restriction site, and the (Gly₄Ser)₃ scFv linker tothe 5′ end of the V_(L) genes (see FIG. 9). VL repertoire DNA was gelpurified, digested with Nco1 and NotI and ligated into NcoI-NotIdigested pYD2 DNA. The ligation mixture was used to transform E. coliDH5α, creating a library that was determined to be diverse by PCRfingerprinting and DNA sequencing. To create light chain shuffled scFvlibraries, light chain library DNA was prepared and digested with eitherNcoI or HindIII and XhoI. It was determined that when cutting withNco1-Not1, recombination could occur between the scFv linker DNA and theGly-Ser linker after the AgaII protein, resulting in approximately 25%of transformants having no light chain. Digesting with HindIII cuts inAgaII, eliminating this problem. The V_(H) gene was amplified from itsrespective scFv gene in pYD2 using primers that had 25 nucleotidesequences complementary to the 5′ and 3′ ends of the digested libraryvector DNA depending on whether the vector was digested with Nco1 orHindIII, respectively. Gel purified V_(H) gene was mixed with digestedvector DNA and used to transform LiAc-treated EBY100 cells. Chainshuffled libraries were created for scFv 4A1.1, 5A20.4, B12.1, 2B18.1,1B10.1, 4B19.1, and 2B25.1.

To select higher affinity scFv, light chain shuffled libraries weregrown and scFv display induced. Yeast were stained with BoNT/A1 orBoNT/B1 at a concentration 10 times greater then the K_(D) and equal tothe K_(D) for the first two rounds of sorting respectively with themajority of BoNT binding yeast collected. Subsequent rounds of sortingwere increasingly stringent with the antigen concentration decreased andless than 1% of the yeast collected. A total of four to six rounds ofsorting were performed for each chain shuffled library, after which thesort output was plated to allow for characterization of individual yeastdisplayed scFv. Twelve individual clones were characterized by DNAsequencing of the scFv gene and the affinity for BoNT determined aspreviously described.

Construction and Purification of IgG

The V_(H) and V_(K) genes of scFv ING1, ING2 were amplified with primersannealing to the 5′ and 3′ ends of the full length V_(H) and V_(K) genesand containing in frame Mlu1 and NheI or DraIII and BsiWI restrictionsites respectively for cloning into N5KG1 Val-Lark or N5LG1Val-Lark (seeNowakowski et al. 2002, supra and Razai et al. 2005, supra for detailsof primer design). These vector results in expression of IgG of theγ1/kappa or γ1/lambda isotype. Amplified V_(H) DNA was digested withMlu1 and NheI, and ligated into N5KG1Val-Lark or N5LG1Val-Lark. Clonescontaining the correct V_(H) were identified by DNA sequencing.Amplified V_(K) genes were cloned into pCR-TOPO vector (Invitrogen) andclones containing the correct V_(K) identified by DNA sequencing. V_(K)genes were excised from pCR-TOPO vector by digestion with DraIII andBsiWI and ligated into DraIII- and BsiWI-digested N5KG1Val-Lark orN5LG1Val-Lark DNA containing the appropriate V_(H) gene. Clonescontaining the correct V_(H) and V_(K) genes were identified by DNAsequencing, and vector DNA was used to transfect CHO DG44 cells byelectroporation. Stable cell lines were established by selection in G418and expanded into 1 L spinner flasks. Supernatant containing IgG werecollected, concentrated by ultra filtration, and purified on Protein G(Pharmacia) column.

Measurement of Solution Phase Affinity at Equilibrium

Equilibrium binding studies were conducted at room temperature (˜25° C.)using a KinExA 3000 flow fluorimeter to quantify the free BoNT/A orBoNT/B at equilibrium using varying concentrations of antibody aspreviously described (Razai et al. 2005, supra). Studies of reactionmixtures were performed in PBS (pH 7.4), with 1 mg/ml BSA and 0.02%(w/v) sodium azide as a preservative. Antibody was serially diluted intoa constant concentration of pure BoNT/A or BoNT/B sufficient to producea reasonable signal, where the antibody concentration was varied fromless than 0.1 to greater than 10-fold above the value of the apparentK_(D). The BoNT/A and BoNT/B concentrations were no more than 4-foldabove the K_(D) to ensure a K_(D) controlled experiment. Samples wereallowed to reach equilibrium for as long as two days, then each of the12 dilutions were passed over a flow cell with a 4 mm column ofAzlactone beads (Sapidyne Instruments) covalently coated with thecorresponding antibody to capture the free BoNT/A or free BoNT/B.Passing an ALEXA FLUOR 647 dye labeled BoNT/A or BoNT/B antibody bindinga non-overlapping epitope over the beads produced a signal relative tothe amount of free BoNT/A or BoNT/B bound to the beads. All data pointswere run in duplicate and sample volume varied from 4 to 25 ml dependingon antibody affinity. The equilibrium titration data were fit to a 1:1reversible binding model using KinExA Pro Software (version 1.0.2;Sapidyne Instruments) to determine the K_(D) (Drake et al. 2004 Anal.Biochem. 325:35-43).

Results

Generation of Human Yeast Displayed scFv Antibody Libraries

Immune yeast displayed scFv antibody libraries were constructed fromhuman volunteers immunized with pentavalent botulinum toxoid (serotypesBoNT/A1, BoNT/B1, BoNT/C, BoNT/D, and BoNT/E). RNA was prepared from theperipheral blood lymphocytes of six different donors and theimmunoglobulin heavy (V_(H)) and kappa light (V_(K)) chain variableregions amplified by using the polymerase chain reaction (PCR) aspreviously described (Marks 1991, supra; Marks et al. J Mol Biol. 1991,222:581-97). V_(H) and V_(K) gene repertoires from each donor werespliced together to create scFv gene repertoires which were cloned fordisplay as N-terminal fusions to the agglutinin receptor (AgaII) proteinon the surface of yeast. A total of six yeast displayed scFv librarieswas generated, ranging in size from 4.1 to 25.7×10⁶ members. Eachlibrary was diverse as determined by PCR fingerprinting and DNAsequencing of 10 randomly selected clones. After induction of scFvdisplay, the percentage of yeast displaying scFv ranged from 45-55% asdetermined by staining with SV5 antibody binding the C-terminal SV5 tagfused to each scFv.

Generation of High Affinity Human Antibodies to Type B BotulinumNeurotoxins

To generate a panel of human antibodies to type B botulinum neurotoxins,six different yeast displayed scFv libraries were sorted separately onBoNT/B1. Sorts were performed using relatively high concentrations ofBoNT/B1 holotoxin in the initial rounds (50-100×10⁻⁹ M, nM) to ensurecollection of all antigen binding scFv. In later rounds, the antigenconcentration was decreased to between 1-10 nM to select for the higheraffinity antibodies and sorts were performed on other BoNT/B subtypes(BoNT/B2, B3, and B4). Libraries were sorted a total of three to sixrounds and yeast displayed scFv from individual colonies were screenedfor binding to BoNT/B1. Antigen binding clones were furthercharacterized with respect to the diversity of scFv present using colonyPCR, BstN1 fingerprinting (Marks et al. 1991, supra), and DNAsequencing. In this manner, 18 scFv were isolated, each with a differentV_(H) CDR3 (Table 8). The equilibrium binding constant for BoNT/B1 wasmeasured for each of the yeast displayed scFv (Table 8). Affinitiesranged from 60 to 0.08×10⁻⁹ M, with a mean K_(D) of 8.6×10⁻⁹ M. For manyof these scFv, a number of additional clonally related scFv were alsoisolated that were of lower affinity.

TABLE 8  Characteristics of lead yeast displayed scFv BoNT/B antibodies.  V_(H)/V_(k) BoNT/B K_(D) bp FACS Gene (x 10⁻⁹ M⁻¹) Clone EpitopeV_(H) CDR3 Sequence Family B1 B2 B3 B4 4B6 L_(C) HDSRYKYFYFGMDV VH5/VKI2.7 1.77 1.08 1.61 (SEQ ID NO: 390) 4B7 L_(C) MSGSRSYSQYYFDS VH4/VK129.4 34.8 25.1 10.0 (SEQ ID NO: 911) 1B10 L_(C) DLTRFHDTTFGVFEM VH3/VKI11.2 9.4 >5000 4.69 (SEQ ID NO: 488) 4B19 L_(C) EWTQLWSPYDY VH1/VKI 6.457.03 3.9 3.3 (SEQ ID NO: 474) 1B22 L_(C) TAFYYENTGPIRCYLDF VH4/VKI 0.520.46 0.39 0.48 (SEQ ID NO: 481) 2B23 L_(C) ALVGRYDISTGYYRPVMDS VH3/VKI0.08 0.13 0.25 0.19 (SEQ ID NO: 524) 2B24 L_(C) DGPMAAIPFYYFDF VH3/VLI0.79 4.0 4.0 0.72 (SEQ ID NO: 531) 2B25 L_(C) GVPIYDSSGTYRGTYFDY VH4/VLI0.95 0.63 0.93 0.68 (SEQ ID NO: 538) 2B27 L_(C) RRLLGPSPYYFDY VH3/VLI1.9 0.56 2.32 0.77 (SEQ ID NO: 558) 2B29 L_(C) GNPQYDTSGSYTGLYFDFVH4/VL1 1.11 1.02 1.61 1.29 (SEQ ID NO: 572) 4B3 H_(N)DILYYHDSSDYWGRGHFYYMDV VH3/VKI 26.7 43.0 1265 NB (SEQ ID NO: 920) 1B11H_(N) DRYPIDCSGGSCFSYGMDV VH3/VK1 2.6 2.1 4.43 NB (SEQ ID NO: 418) 1B18H_(N) LEWGGRNGWVSP VH3/VKI 7.3 3.8 3.04 1.7 (SEQ ID NO: 460) 4B1 H_(C)DKRTYEYNWNSLWF VH1/VK1 1.08 14.9 NB 0.85 (SEQ ID NO: 383) 4B5 H_(C)MRGYSSWHYSYYYVMDV VH3/VKI 59.6 NB NB NB (SEQ ID NO: 924) 1B12 H_(C)DRSHYGDYVGYLDY VH3/VKI 1.17 1.08 NB 6.01 (SEQ ID NO: 439) 1B14 H_(C)SSIVGAPYGMDV VH3/VKI 0.98 >5000 NB 2.12 (SEQ ID NO: 926) 2B30 H_(C)DVSEYGDYVGHFDY VH3/VL1 0.37 0.19 NB NB (SEQ ID NO: 579) Clone name,epitope, V_(H) CDR3 sequence, V_(H) and V_(κ) germline gene family andequilibrium dissociation constant (K_(D)) for BoNT/B subtypes are shown.scFv K_(D) measured on yeast displayed scFv. L_(C) = toxin light chain;H_(N) = toxin translocation domain; H_(C) = toxin binding domain; NB =no binding.

To determine the BoNT/B subtype specificity, the K_(D) of the yeastdisplayed scFv were measured for BoNT/B2, B3, and B4 (Table 8). A numberof scFv only bound the immunizing BoNT/B1 subtypes, e.g. mAb 4B1. Todetermine which BoNT/B functional domain the scFv bound, the BoNT/BH_(C), H_(N), and L_(C) genes were cloned into pYD2 and displayed on thesurface of Saccharomyces cerevisiae (FIG. 6), as previously reported forBoNT/A domains (Levy et al. J Mol Biol. 2007, 365:196-210). Each domainwas well displayed on the yeast surface, as quantitated using aC-terminal SV5 tag fused to each domain (FIG. 6). The domain recognizedby each of the scFv was determined by incubating yeast displayed BoNT/Bdomains with either native scFv expressed in E. coli or IgG constructedfrom scFv genes (see below). Native scFv was generated by subcloning thescFv genes into the bacterial secretion vector pSYN1. To determine howmany non-overlapping BoNT/B epitopes were recognized by the leadantibodies, yeast displayed scFv were incubated with BoNT/B holotoxin,followed by incubation with purified native scFv. scFv recognizingoverlapping epitopes showed no yeast staining while scFv bindingnon-overlapping epitopes stained the yeast surface. For some of theseassays, purified IgG constructed from the scFv V-genes was used foryeast staining instead of the scFv. Using these assays, it wasdetermined that of the 18 scFv, five bound the BoNT/B H_(C), three boundthe H_(N), and ten bound the L_(C) (FIG. 7). A total of ninenon-overlapping epitopes were recognized by the scFv antibodies, threeon the H_(C), two on the H_(N), and four on the L_(C). Seven of the tenL_(C) antibodies clustered within one antibody footprint of each other(FIG. 7). Of note, of the 10 scFv that bound to all four BoNT/Bsubtypes, 9 bound to L_(C), 1 bound to H_(N), and none bound to H_(C).This is consistent with the relative percent homology of the threedomains between the four BoNT/B subtypes, the L_(C) is the mostconserved and the H_(C) is the least conserved.

Generation and Characterization of High Affinity Human Antibodies toType A Botulinum Neurotoxins

The sortings of yeast displayed scFv libraries were designed to generateantibodies that bound multiple BoNT/A subtypes and/or bound to epitopesnot recognized by previously generated antibodies. To generateantibodies binding multiple BoNT/A subtypes, a library constructed fromdonor six was sequentially sorted on BoNT/A1 and BoNT/A2 (FIG. 8). Togenerate antibodies binding epitopes not recognized by existing mAbs,yeast libraries were incubated with BoNT/A then simultaneously stainedwith mAbs 3D12 and AR2 labeled with different fluorophores and sorted(FIG. 8). Using this approach, two yeast displayed scFv were identified,ING1 and ING2. A number of additional scFv were also isolated that wereof lower affinity but which were clonally related to ING1 or ING2,having the same V_(H) CDR3 but point mutations in the V_(H) gene and/ordifferent light chain genes. ING1 and ING2 both bound BoNT/A1 and A2with high affinity as yeast displayed scFv, but only ING1 bound BoNT/A3(Table 9). Additional mAbs to BoNT/A epitopes were generated by sortinga yeast displayed library constructed from donor ten on BoNT/A1. Fromthese sorts, two new yeast displayed scFv were identified, 4A1 and 5A20.Both of these yeast displayed scFv only bound BoNT/A1. A number ofadditional scFv were also isolated that were of lower affinity but whichwere clonally related to 4A1 or 5A20, having the same V_(H) CDR3 butpoint mutations in the V_(H) gene and/or different light chain genes.

TABLE 9  Characteristics of lead yeast displayed BoNT/A antibodies.V_(H)/V_(k) BoNT/A K_(D) bp FACS Epi- V_(H) CDR3 Gene (x 10⁻⁹ M⁻¹) Clonetope Sequence Family A1 A2 A3 ING2 L_(C) DPYYYSYMDV VH1/VK4 0.24 0.25 NB(SEQ ID NO: 867) 5A20 L_(C) EASFGWSYLG VH1NK1 0.40 NB NB HDDAFDI (SEQ IDNO: 869) 4A1 H_(N) DPGWIYSDTSP VH3/VK1 7.4 >5000 NB AAGWFD (SEQ IDNO: 871) ING1 L_(C)-H_(N) VRTKYCSSLSC VH3/VK1 5.28 3.83 ND FAGFDS(SEQ ID NO: 884) Clone name, epitope, VH CDR3 sequence, V_(H) and V_(κ)germline gene family and equilibrium dissociation constant (K_(D)) forthree BoNT/A subtypes are shown. scFv K_(D) measured on yeast displayedscFv. L_(C) = light chain; H_(N) = translocation domain; L_(C)-H_(N) =epitope requiring presence of both L_(C) and H_(N) for binding; NB = nobinding; ND = not determined due to absence of purified BoNT/A3.

Yeast displayed BoNT/A scFv were further characterized to determinewhich BoNT domain they bound by staining with recombinant BoNT/A H_(C),H_(N), or L_(C). 4A1 was determined to bind the H_(N) domain and bothING2 and 5A20 bound the L_(C) (Table 9). We previously determined usingyeast displayed BoNT/A domains that ING1 recognized a complex epitoperequiring the presence of both the N-terminal domain of the BoNT/A heavychain translocation domain (H_(N)) and the BoNT/A light chain by (L_(C))(Levy et al. 2007, supra). To determine whether the epitopes recognizedby the antibodies overlapped, yeast displayed ING1, ING2, 4A1, or 5A20scFv was incubated with BoNT/A and then stained with IgG constructedfrom each of these scFv. Each of these mAbs was found to recognizenon-overlapping epitopes.

Affinity Maturation of Antibodies to Type A and Type B BotulinumNeurotoxins

Since it is statistically improbable that the original immune cognateV_(H)-V_(L) pair is recreated in the primary library, scFv affinitymaturation was performed by recombining the scFv V_(H) gene with alibrary of V_(L) genes, so called light chain shuffling (Clackson et al.Nature 1991, 352:624-8; Marks et al. Biotechnology (N Y). 1992,10:779-83; Schier et al. J Mol Biol. 1996, 263:551-67). To facilitatelight chain shuffling, a light chain library was created in the yeastdisplay vector pYD2 by subcloning the V_(L) genes from the donor six,nine, and ten scFv gene repertoires in pYD2. To further increase lightchain diversity, especially since lambda light chain V-genes had notbeen amplified from the immune donors, the light chain repertoire from alarge non-immune scFv phage antibody library (Sheets et al. 1998, supra)that had been subcloned into pYD2 was also utilized. V_(L) genes wereamplified from the scFv gene repertoires in pYD2 by using PCR and thenreamplified to append the 3′ end of the V_(H) framework 4 (FR4),containing a Xho1 restriction site, and the (Gly4Ser)3 scFv linker tothe 5′ end of the V_(L) genes (FIG. 9). After cloning into pYD2, alibrary of size 4.2×10⁷ was created that was determined to be diverse byPCR fingerprinting and DNA sequencing.

To create light chain shuffled scFv libraries, the light chain librarywas digested with NcoI or HindIII and XhoI. The V_(H) gene was amplifiedfrom its respective scFv gene in pYD2 using primers that had 25nucleotide sequences complementary to the 5′ and 3′ ends of the digestedlibrary vector DNA and was cloned into Saccharomyces cerevisiae by gaprepair. Alternatively, scFv chain shuffled libraries were created byamplifying the V_(H) FR4-scFv linker-V_(L) gene repertoire from pYD2 andsplicing it to a specific V_(H) gene by overlap extension. The chainshuffled scFv gene repertoire was then cloned into pYD2. A total ofseven chain shuffled libraries were created from the V_(H) genes of scFv4A1, 5A20, 1B18, 1B10, 1B22, 2B25, and 4B19. Their sizes ranged from2.0×10⁷ to 4.0×10⁷. An additional chain shuffled library was createdfrom the V_(H) genes of scFv B12 (see table 3) using splicing by overlapextension as previously described (Marks et al. 1992, supra). Each ofthese libraries was sorted separately using BoNT/A1 or BoNT/B1 atdecreasing concentrations for four to six rounds until a single clonebecame dominant. The highest affinity clone was identified by DNAsequencing and measuring the K_(D) of yeast displayed scFv. The K_(D) ofthe light chain shuffled scFv increased 2.2 to 74 fold from the K_(D) ofthe parental scFv to a K_(D) between 1.0 and 6.38×10⁻¹⁹ M. In general,the largest increase in affinities was observed for the lowest affinityparental scFv. The average K_(D) increased 16 fold from 4.98×10⁻⁹ M to0.31×10⁻⁹ M (Table 10).

TABLE 10 Affinities of affinity matured scFv and IgG for BoNT/A andBoNT/B subtypes. scFv K_(D) measured on yeast displayed scFv. IgG K_(D)measured in solution using a flow fluorimeter (KinExA). ScFv AffinityFold K_(D) increase in K_(D) of affinity matured IgG for BoNT Clone(×10⁻¹²M⁻¹) affinity scFv by KinExA (×10⁻¹²M⁻¹) Initial K_(D) wildIsolate/Affinity type/K_(D) matured BoNT/A1 matured A1 A2 A3 4A1/4A1.1100 74 11.34 >1000 NB 5A20/5A20.4 182 2.2 13.6 NB NB BoNT/B1 B1 B2 B3 B4B12/B12.1 300 30 33.0 16.2 46.2 625 1B18/2B18.1 638 11.4 72.4 181 98.4382 1B10/1B10.1 235 47.7 0.33 0.31 1200 0.47 2B25/2B25.1 349 2.7 16.753.2 17.7 30.0 4B19/4B19.1 396 25 176 138 156 194

Impact of Conversion of Yeast Displayed scFv to IgG on Affinity

For many applications, such as diagnostic ELISA based assays as well asin vivo BoNT neutralization studies, it is desirable to utilize IgG. Todetermine the success rate of converting scFv to IgG, we convertedtwelve yeast displayed scFv, including five lead antibodies and sevenaffinity matured chain shuffled scFv, to full length IgG consisting ofthe human gamma 1 constant region and the human kappa or lambda constantregion (Nowakowski et al. 2002, supra). Stable CHO DG44 cell lines wereestablished for each of the twelve antibodies and IgG was purified fromcell culture supernatant. The monovalent K_(D) was determined for eachIgG using kinetic exclusion analysis (KinExA).

Each of the twelve scFv was successfully cloned, stable cell linesestablished, and IgG purified at yields of 5 to 50 mg/L of cell culturesupernatant. The solution K_(D) of each of these IgG was lower (higheraffinity) than the parental scFv (Tables 10, and 11). The affinity ofthe five IgG constructed from the lead scFv increased 1.5 to 25 foldfrom the affinity of the parental scFv, with the average K_(D)decreasing 8.8 fold from 2.68×10 M to 0.31×10⁻⁹ M (Table 11). Similarly,the affinity of the seven IgG constructed from the affinity matured scFvincreased 2.25 to 712 fold from the affinity of the parental scFv, withthe average K_(D) decreasing 6.43 fold from 3.14×10⁻¹⁰ M to 0.49×10⁻¹⁰ M(Table 10). Six of the seven IgG constructed from the affinity maturedscFv had K_(D)'s less than 1.0×10⁻¹⁰ M.

TABLE 11 Affinities of lead scFv and IgG for BoNT/A and BoNT/B subtypes.scFv Affinity IgG Affinity for BoNT by KinExA Clone K_(D) (×10⁻¹²M⁻¹)K_(D) (×10⁻¹²M⁻¹) BoNT/A1 A1 A2 A3 ING1 5280 314.3 719.1 400 ING2 2389.57 7.42 NB BoNT/B1 B1 B2 B3 B4 1B18 7300 816 817 972 21 1B22 515 335319 221 129 2B23 79 38.2 45.3 48.0 55.2 scFv K_(D) measured on yeastdisplayed scFv. IgG K_(D) measured in solution using a flow fluorimeter(KinExA). NB = no binding.Discussion

These studies indicated that the scFv were diverse with respect toepitope recognized and were of an affinity (average K_(D)=9.2×10⁻⁹ M)expected from an immune library. On average, three lead scFv weregenerated from each library constructed from a human donor, a relativelysmall number.

The magnitude of the increase in affinity from chain shuffling did notappear to be dependent on whether the source of the V_(L) genes was fromthe same donor as the initial V_(H)-V_(L) gene pairing or not.Presumably, light chain diversity across donors was similar enough thatshuffling recapitulated the high affinity scFv binding site. The averageaffinity of the seven IgG constructed from the chain shuffled scFvV-genes was 45 pM, a value that would be at the very high end ofantibodies generated by the humoral immune system. Three of theseantibodies had K_(D)<15 pM. The very high average affinity suggests thatthese scFv do not represent the cognate V_(H)-V_(L) pairing, whichshould not have such a high affinity. Rather it is likely that a V_(L)pair was found for the V_(H) that generated an antibody of higheraffinity than that of the cognate V_(H)-V_(L) pairing.

Example 2 Affinity Maturation of Human Botulinum Neurotoxin Antibodiesby Light Chain Shuffling Via Yeast Mating

The following methods and materials were used in the present example.

Methods and Materials

Oligonucleotide primers Primers for V_(L) library construction(SEQ ID NO: 908) For: 5P-AAGGCTCTTTGGACAAGAGAAACTCTGGATCCVH specific forward oligo (SEQ ID NO: 909)Rev: 5P-GTGCCAGGGGGAAGACCGATGGGCCCTTGGTGCTAGC VH specific reverse oligo

Strains, Media Antibodies, and Toxins:

Yeast strains Saccharomyces cerevisiae JAR300 ((GAL1-AGA1TURA3 ura3-52trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 MATa) and YVH10 (BJ5464Ura-Trp-MAT alpha) were maintained in synthetic dextrose plus caseinamino acids (SD-CAA, 0.7% yeast nitrogen base, 0.1M Sodium phosphate,0.5% casamino acids, 2% dextrose, 0.006% leucine) media with tryptophanand uracil added. After transformation with pPNL20s, JAR300transformants were selected in SD-CAA media with uracil. Aftertransformation with pPNL30s, YVH10 transformants were selected in SDCAAmedia with tryptophan. Yeast mating was performed on YPD plates (CurrentProtocols in Molecular Biology, John Wiley and Sons, Chapter 13.1.2).After mating, diploid yeast were selected on SD-CAA media. Fab surfacedisplay was induced by transferring yeast cultures from SD-CAA to SG-CAAmedium (identical to SD-CAA medium except the glucose was replaced bygalactose) and growing at 18° C. for 24-48 hours. Bacteria strain E.coli DH5α, (K12, Δ(lac-pro), supE, thi, hsdD5/F′ traD36, proA+B+, lacIq,lacZΔM15) was used for all cloning and preparation of plasmid DNA.Chinese Hamster Ovary cell line CHO DG44 was maintained in CHO-SFM-IImedia (Invitrogen). Pure BoNT/A1 (Hall hyper), BoNT/A2 (FRI-H1A2),BoNT/B1, BoNT/B2, BoNT/B3, and BoNT/B4 were purified from theirrespective strains or purchased from Metabiologics (Madison, Wis.).Complex BoNT/A3 was expressed and purified from strains A254 and Ba207.Mouse anti-SV5 antibody was purified from hybridoma supernatant usingProtein G and directly labeled with ALEXA FLUOR 488 dye or ALEXA FLUOR647 dye using a kit provided by the manufacturer (MOLECULAR PROBES).Chinese ovary cells (CHO) supernatants (Nowakowski et al. 2002, supra;Razai et al. 2005, supra). For flow cytometry (FACS), purified human ormouse IgGs were directly labeled with either ALEXA FLUOR 647 dye orALEXA FLUOR 488 dye using a kit provided by the manufacturer (MOLECULARPROBES).

Construction of a Light Chain Repertoire in YVH10 for Light ChainShuffling (Donor 9)

A single light chain repertoire in YVH10 was constructed containingkappa and lambda light chains. First, vector pPNL30 was modified toinsert a 90 base pair stuffer between the Xho1 and BsiWI sites.

Two different methods were used to construct the three Fab libraries foraffinity and specificity maturation of three scFv lead clones: ING1, B6and B11. For ING1 and B6 Fab library construction before yeast mating, asingle VH gene from the lead scFv was PCR amplified using the specificprimers mentioned above. But for B11Fab library construction,error-prone PCR was applied when preparing a mutated VH repertoire fromthe lead scFv B11 VH gene. The single VH gene of ING1 or B6, or thewhole repertoire of randomly mutated B11 VH gene were then cloned intopPNL20S vector. Cloning was achieved by co-transforming JAR300 yeast (bythe method of Gietz and Schiestl) with BamH I/Nhe I linearized vectorand gap-tailed amplicon utilizing yeast gap repair. The transformantswere selected on SD-CAA+uracil before mating with VL library yeast.

The VL libraries were constructed using conventional methods. Briefly,the light chain vector pPNL30S was digested with XhoI/BsiWI andgel-purified. YVH10 yeast was used for pPNL30S+VL transformation, andthe transformants selected on SD-CAA+tryptophan.

For yeast mating, fresh cultures of VL library in YVH10/pPNL30-LC (MAT αstrain) after selection were grown in the selective media at 30° C. withshaking, and a single VH clone or a repertoire of VH (for B11) libraryin JAR300/pPNL20-HC (MAT “a” strain) were grown in another selectablemedia similarly. 1 OD₆₀₀/ml (2×10⁷ yeast) of each culture were mixedtogether, pelleted, and resuspended in 200 μl YPD media before placingin the center of a pre-warmed 30° C. YPD plate without spreading. Theplates were incubated at 30° C. for 6 h for yeast mating and the yeastwere resuspended in SD-CAA medium from the plate after mating. To checkthe mating efficiency and the final Fab library size, appropriatedilutions of mated yeast were plated on SD-CAA agar plates and the Fablibrary was selectively grown in SD-CAA media without tryptophan oruracil. The OD₆₀₀ reading at the start of growth was 0.025 to allowgrowth of the diploids to outcompete the non-growing haploids.

For diversity analysis, ten randomly picked clones from each librarywere checked by BstNI fingerprinting and/or sequencing for both VH andVL genes.

For Fab expression induction, freshly saturated SD-CAA cultures of theyeast libraries were induced in SG-CAA liquid media at 18° C. for 24-48h with shaking. Anti-SV5/ALEXA FLUOR 647 dye were used to check the Fabexpression after induction.

Selection of Mutant Clones from Fab Libraries by FACS

The optimally expressed diploid yeast were FACS sorted two rounds withone of the subtypes of BoNT/A or B, then using different subtypes ofBoNT/A or B for the following rounds of sorting. An amount of diploidyeast at least ten times larger than the library size or the sort outputfrom the previous round were washed and resuspended in FACS buffer, anda desired concentration of one subtype of pure BoNT/A or B was added.Sorting and selections were generally performed after allowing thereaction mixture to come to equilibrium. The volume for incubation ofyeast with toxin was chosen to ensure that toxin was in at least a fivefold excess over the number of expressed Fab (assuming 5×10⁵ Fab/yeast).Incubation times were chosen to ensure that the reaction had come to atleast 90% of equilibrium, using the formula shown in the Discussionsection below. BoNT/A1 concentration for the first round of sorting ofthe ING1 Fab library was 10 nM. The 1^(st) round sort output weredivided into two parts after yeast growth and induction for the secondround sorting: one with 10 nM BoNT/A2, the other using 5 nM BoNT/A1.Since both BoNT/A1 and A2 binders are clearly enriched after two roundsof sorting, for better cross reactive and higher affinity cloneselection, decreased concentrations of BoNT/A2 were used for thefollowing 3 rounds of sorting: 5, 1 and 0.5 nM before individual cloneswere picked out for sequencing and affinity measurement. BoNT/B 1concentrations used for the 5 rounds of sorting of the B6 Fab librarywere: 100, 25, 10, 5, or 2 nM separately, while those for the B11 Fablibrary were: 25, 10, 2, 1, or 1 nM. After incubation, cells were washedwith ice-cold FACS buffer and resuspended in a 1:400 dilution of Alexa488 labeled anti-SV5 antibody and one of the following: ALEXA FLUOR 647dye labeled 7C1 or 3D12 antibody (for the ING1 Fab library), or twoALEXA FLUOR 647 dye labeled anti-BoNT/B mAb (A12 and 6A12), which boundthe catalytic domain of the toxin, an epitope that did not overlap withB6 or B11 (for the B6 Fab library and B11 Fab library). Cells wereincubated for 30 minutes with secondary antibodies at 4-8° C., washedonce with FACS buffer, resuspended in 0.5-1 ml of FACS buffer and sortedon a FACSAria. Typically 0.5-5% of the expressing and toxin bindingpopulation was gated for collection in the first 2 round sorting, and0.1˜0.5% of those populations was gated in the following rounds sorting.Collected cells were grown in SD-CAA media and used for the next roundof sorting after induction in SG-CAA as described above.

Over a period of 2-3 weeks, each library was sorted 4-6 rounds withsequentially diluted different subtypes of BoNT A or B. Ninety-sixindividual clones were picked out after the last round of sorting, and500 pM ALEXA FLUOR 647 dye or ALEXA FLUOR 488 dye labeled toxin wereused to screen the best candidates in terms of expression and binding byFACS analysis. Using MFI as a single criterion after fixed concentrationtoxin staining, six to twelve clones were chosen from the screened onesfor further K_(D) measurement and sequence analysis.

Quantitative equilibrium binding was determined using flow cytometry. Ingeneral, six different concentrations of one subtype of pure BoNT/A or Bwere utilized spanning a range of concentrations from ten times above toten times below the K_(D). Incubation volumes and number of yeaststained were chosen to keep the number of antigen molecules in tenfoldexcess above the number of expressed Fab, assuming 5.0×10⁵ Fab/yeast.Incubation times were chosen based on anticipated times to equilibriumcalculated using approximations of the anticipated k_(on) and k_(off)(see above). This was usually performed by overnight incubation. Bindingof either subtype of BoNT/A to yeast-displayed Fab was detected using a1:200 dilution of 1 mg/ml monoclonal antibody binding a non-overlappingBoNT/A epitope (3D12 for the ING1 Fab library)labeled with ALEXA FLUOR647 dye. To quantify the protein-ligand affinity constant (K_(D)) withinthe surface display context, only the Fab displaying yeast (SV5 bindingpositive) were included in the analysis by co-staining with SV5-ALEXAFLUOR 488 dye. Each K_(D) was determined in triplicate, three separateinductions and measurements. The best Fab clone from the ING1 Fablibrary was designated as 2G11, the highest affinity clone from B6 Fablibrary was named as B6.1, and that from the B11 Fab library was calledB11E8 (Table 13).

Generation of IgG from Fab

IgG were generated from Fab genes. Briefly, the VH genes were amplifiedusing PCR from their respective pPNL20 vectors with the primer pairs.DNA was digested with MluI and NheI, ligated into N5KG1Val-Lark (a giftfrom Mitch Reff, IDEC Pharmaceuticals, San Diego) and clones containingthe correct VH identified by DNA sequencing. Vk genes were amplifiedfrom the same diploid yeast containing pPNL30 vectors and cloned intopCR-2.1 vector (Invitrogen) and clones containing the correct Vkidentified by DNA sequencing. Vk genes were excised from pCR-2.1 vectorwith DraIII and BsiWI and ligated into DraIII and BsiWI-digestedN5KG1Val-Lark DNA containing the appropriate VH gene. Clones containingthe correct VH and Vk gene were identified by DNA sequencing, and vectorDNA was used to transfect CHO DG44 cells by electroporation. Stable celllines were established by selection inG418 and expanded into one literspinner flasks. Supernatant containing IgG was collected and purified onProtein G (Pharmacia). IgG purity was assessed by native and denaturingSDS PAGE and concentration determined by A₂₈₀ nm.

Measurement of IgG Solution Equilibrium Binding Constants by FlowFluorimetry

Equilibrium binding studies were conducted at room temperature (˜25° C.)using a KinExA 3000 flow fluorimeter to quantify the free BoNT/A or B atequilibrium using varying concentrations of antibody. Studies ofsensitive detection of BoNT/A or B reaction mixtures were performed inPBS (pH 7.4), with 1 mg/ml BSA and 0.02% (w/v) sodium azide as apreservative. Antibody was serially diluted into a constantconcentration of BoNT/A or B sufficient to produce a reasonable signal,where the antibody concentration was varied from less than 0.1 togreater than tenfold above the value of the apparent K_(D). The BoNT/Aor B concentrations were no more than fourfold above the K_(D) to ensurea K_(D) controlled experiment. Samples were allowed to reach equilibriumfor as long as two days, then each of the 12 dilutions were passed overa flow cell with a 4 mm column of Azlactone beads (Sapidyne Instruments)covalently coated with the corresponding antibody to capture the freeBoNT/A or B. Passing an Alexa-647 labeled BoNT/A or B antibody binding anon-overlapping epitope over the beads produced a signal relative to theamount of free BoNT/A or B bound to the beads. All data points were runin duplicate and sample volume varied from 4 to 25 ml depending onantibody affinity. The equilibrium titration data were fitted to a 1:1reversible binding model using KinExA Pro Software (version 1.0.2;Sapidyne Instruments) to determine the K_(D).

Antibodies Selected for Affinity Maturation by Light Chain Shuffling

One BoNT/A and two BoNT/B scFv antibodies were chosen for affinitymaturation. The BoNT/A scFv antibody ING1 was selected from an immunescFv yeast display library constructed from a human donor (donor 6)immunized with pentavalent botulinum toxoid. ING1 binds the BoNT/A1translocation domain (H_(N)) with an equilibrium dissociation constant(K_(D)) of 5.28×10⁻⁹ M as a yeast displayed scFv (Table 12). Two BoNT/BscFv antibodies (B6 and B11) were also selected from an immune scFvyeast display library constructed from a human donor immunized withpentavalent botulinum toxoid. B6 binds the BoNT/B light chain (L_(C))with a K_(D) of 2.71×10⁻⁹ M and B11 binds the BoNT/B H_(N) with a K_(D)of 2.62×10⁻⁹ M (Table 12).

TABLE 12 Equilibrium dissociation constants for wild-type and affinitymatured BoNT antibodies. Equilibrium dissociation constants (K_(D)) forwild type and affinity matured yeast displayed BoNT antibodies in boththe scFv and Fab format were measured by flow cytometry. Clone InitialAffinity BoNT K_(D) by FACS Isolate matured (×10⁻¹²M⁻¹) scFv Fab scFvFab scFv Fab BoNT/A1 BoNT/A2 ING1 5284 20630 3834 14297 2G11 369 205 205193 BoNT/B1 BoNT/B2 B6 2714 NM 1767 NM B6.1 291 286 394 246 B11 2620 NM2114 NM B11E8 193 81 189 166

Generation of BoNT Fab from the V-Genes of scFv by Yeast Mating

To determine the feasibility of affinity maturing ING1, B6, and B11 byyeast mating, yeast displayed Fab were constructed and the display leveland K_(D) for BoNT determined. The V_(H) gene from each of the threescFv was PCR amplified and cloned directly into S. cerevisiae strainJAR300 by gap repair into BamH I and Nhe I digested pPNL20s. This fusesthe V_(H) gene to the C_(H)1 gene which is fused to the N-terminus ofthe Aga2 yeast surface protein, resulting in display of the V_(H)-C_(H)1on the yeast surface. The V_(k) gene from each of the three scFv was PCRamplified and cloned directly into S. cerevisiae strain YVH10 by gaprepair into XhoI and BsiWI I digested pPNL30s. This fuses the V_(k) geneto the C_(k) and results in secretion of the light chain. To create Fab,S. cervisiae JAR300 (a-mating type) containing the relevant V_(H)-C_(H)1in pPNL20s was mixed with S. cervisiae YVH10 (a-mating type) containingthe relevant light chain in pPNL30s and the resulting diploid yeastselected on uracil⁻, tryptophan⁻ plates. Fab display was induced and thedisplay level and affinity for BoNT measured by flow cytometry. Allthree Fab were well displayed on the yeast surface (data not shown). Theaffinity of the ING1 Fab was 3.9 and 4.4 fold lower than the scFv forBoNT/A1 and BoNT/A2 respectively, while the affinity of the B6 Fab wasessentially identical to the affinity of the scFv for BoNT/B1 andBoNT/B2 (Table 12). Interestingly, the affinity of the B11 Fab was 2.5lower than the scFv for BoNT/B1 but 1.46 higher than the scFv forBoNT/B2 (Table 12). The results indicate that Fab can be constructedfrom the V-genes of scFv using yeast mating and that the K_(D) of yeastdisplayed Fab and scFv are comparable.

Generation and Sorting of Fab Chain Shuffled Libraries

The following strategy was used to create ING1, B6, and B11 light chainshuffled libraries. A library of human kappa and lambda light chainsfrom donor 10 was cloned directly into vector pPNL30s in S. cerevisiaestrain YVH10 by gap repair. The resulting light chain library containedtransformants containing a light chain insert and was diverse asdetermined by DNA sequencing. To create Fab light chain shuffledlibraries, S. cervisiae JAR300 (a-mating type) containing the relevantV_(H)-C_(H)1 (ING1, B6, or B11) in pPNL20s was mixed with S. cervisiaeYVH10 (a-mating type) containing the light chain library in pPNL30s andthe resulting diploid yeast selected on uracil⁻, tryptophan⁻ plates. Thenumber of diploid yeast colonies was at least 100 times greater than thesize of the light chain shuffled library, suggesting that the lightchain library diversity was captured in the chain shuffled library.Analysis of 10 colonies from each mating indicated that each had theexpected wild-type V_(H) gene and a different V_(L) gene. Each of the 10colonies also expressed a Fab on the yeast surface, as determined bystaining with anti-SV5 antibody.

Higher affinity chain shuffled Fab were isolated by FACS. For each ofthe three chain shuffled libraries, Fab expression was induced and yeaststained with 1˜10×10⁻⁸ M (10 nM˜100 nM) BoNT/A1 or BoNT/B1 for the firsttwo rounds of sorting with the majority of BoNT binding yeast collected.Subsequent rounds of sorting were increasingly stringent with theantigen concentration decreased and less than 1% of the yeast collected.A total of six rounds of sorting were performed for each Fab library,after which the sort output was plated to allow for characterization ofindividual yeast displayed Fab. Ninety-six clones were randomly pickedinto 96 well microtiter plates from each of the three Fab sortings andFab expression and BoNT binding quantitated using a single antigenconcentration (5.0×10⁻¹⁰ M, 500 pM). While almost 100% of the pickedclones showed a positive BoNT binding signal, the mean fluorescenceintensity (MFI) varied significantly among them. Twelve individualclones with the highest MFI were chosen for further analysis.

Characterization of Chain Shuffled Fab

The V_(H) and V_(L) genes were PCR amplified from the three sets oftwelve yeast colonies and the V_(H) and V_(L) genes sequenced. For eachof the three chain shuffled libraries, sequence analysis revealed asingle V_(H) gene (the same as the original V_(H) gene) paired withdifferent V_(L) genes (Table 13). Affinities (K_(D)) of the yeastdisplayed Fab were measured by flow cytometry for BoNT/A1 or BoNT/B1 andcompared to the affinities of the parental scFv (Table 13). Overall theaffinities of the best ING1, B6, and B11 chain shuffled Fab increased 3to 43 fold compared to their parental scFv, with yeast displayed K_(D)ranging from 32 to 1645 pM (Table 13). The complete amino acid sequencesof the V_(H) and V_(L) of the antibodies described in Table 13 areprovided in FIG. 11.

TABLE 13  Characteristics of affinity matured BoNT antibodies. Number ofmutations Amino acid sequence of from Germline BoNT K_(D) K_(D) wildClone Vk CDRs wild-type family by FACS type/K_(D) Name CDR1 CDR2 CDR3Vk gene gene (x 10⁻¹² M⁻¹) matured BoNT/A1 ING1 RASQSISSYLN AASSLQSQQSYSTPRTT 0 IGKV1- 5284 (SEQ ID (SEQ  (SEQ ID 39*01 NO: 1042) ID NO:NO: 1044) 1046) 2G11 ----------H D---S-- ------RAL- 12 IGKV1- 205 25(SEQ ID (SEQ  (SEQ ID 39*01 NO: 1056) ID NO: NO: 1060) 1058) 1D11----------H D------ ------RAL- 12 IGKV1- 420 12 (SEQ ID (SEQ  (SEQ ID39*01 NO: 1056) ID NO: NO: 1060) 1051) ING1.1C1 ----------- --------------P-- 6 IGKV1- 890  6 (SEQ ID (SEQ  (SEQ ID 39*01 NO: 1049) ID NO:NO: 711) 1044) ING1.2B10 ----------H D---S-- ------RAL- 13 IGKV1- 1645 3 (SEQ ID (SEQ  (SEQ ID 39*01 NO: 1056) ID NO: NO: 1060) 1058) ING1.5B1----------H D---S-- ------RAL- 12 IGKV1- 290 18 (SEQ ID (SEQ  (SEQ ID39*01 NO: 1063) ID NO: NO: 1060) 1065) ING1.3C2 ------G-SNA ----T--------LMCS 16 IGKV1- 710  7 (SEQ ID (SEQ  (SEQ ID 27*01 NO: 1070) ID NO:NO: 1074) 1072) 5G4 ----G--N--A ---T--- ------LMCS 14 IGKV1- 430 12(SEQ ID (SEQ  (SEQ ID 27*01 NO: 1070) ID NO: NO: 1074) 1072) BoNT/B1 B6QAGQDISNFLN DASNLET QQYDNLPYT 0 IGKV1- 2714 (SEQ ID (SEQ  (SEQ ID 33*01NO: 637) ID NO: NO: 641) 639) B6.1 R-S-S--SY-- S--S-QS --SYST-PYT 26IGKV1- 286  9 (SEQ ID (SEQ  (SEQ ID 39*01 NO: 644) ID NO: NO: 648) 646)B6.C12 R-S-S--SY-- A--S-QS --SYTA-C- 24 IGKV1- 405  7 (SEQ ID (SEQ (SEQ ID 39*01 NO: 1238) ID NO: NO: 1242) 1240) B6.D2 R-S-S-RDY-S S--S-QSLEKYSF-RCT 39 IGKV1- 254 11 (SEQ ID (SEQ  (SEQ ID 6*01 NO: 1245) ID NO:NO: 1249) 1247) B11 RASQSINSWLA EASSLES QQYDSYWLT 0 IGKV1- 2620 (SEQ ID(SEQ  (SEQ ID 5*03 NO: 665) ID NO: NO: 669) 667) B11.A5 ----GVSR---G----Q- -----FP-- 16 IGKV1- 61 43 (SEQ ID (SEQ  (SEQ ID 12*01 NO: 1266)ID NO: NO: 1270) 1268) B11.E8 -----VSKF-- G--TRAT ----NWPI- 32 IGKV3 3232 (SEQ ID (SEQ  (SEQ ID D- NO: 679) ID NO: NO: 683) 15*01 681) B11.E9-----VSKF-- G--TRAT ----NWPI- 32 IGKV3 69 38 (SEQ ID (SEQ  (SEQ ID D-NO: 1273) ID NO: NO: 1277) 15*01 1275) B11.F7 -----VG-T-- G--TRAT---N-WPI- 33 IGKV3 177 15 (SEQ ID (SEQ  (SEQ ID D?- NO: 1280) ID NO:NO: 1284) 15*01 1282) B11.H12 ----G--NY-- A--T-Q- ---Y-P-- 19 IGKV1- 17515 (SEQ ID (SEQ  (SEQ ID 8*01 NO: 1287) ID NO: NO: 1291) 1289) Clonename, location of mutations in the Vκ complementarity determiningregions (CDRs), number of mutations between the wild type and affinitymatured Vκ and the germline gene family and germline gene of origin areindicated. Equilibrium dissociation constant (K_(D)) was measured byflow cytometry.

In the case of the BoNT/A antibody ING1, seven Fab were identified whichhad affinities 3 to 25 fold higher than the parental ING1 scFv (Table13). All seven had V_(L) genes derived from the same Vk1 germline genefamily and 5 of the 7 were derived from the same IGKV1-39*01 germlinegene. Compared to the parental Vk gene, the Vk genes from the affinitymatured Fab had 6 to 16 amino acid substitutions (Table 13). In the caseof the BoNT/B antibody B6, three different Fab were identified which hadaffinities higher than the parental B6 scFv (Table 13). All three hadV_(L) genes derived from exactly the same Vk1 germline gene family asthe parental Vk gene. The Vk genes from the affinity matured B6 Fab weremore mutated than those of the ING1 affinity matured Fab, having 26 to39 amino acid substitutions compared to the parental Vk gene (Table 13).In the case of the BoNT/B antibody B11, different Fab were identifiedwhich had affinities 61 to 177 fold higher than the parental B11 scFv(Table 13). Two had V_(L) genes derived from the same Vk1 germline genefamily as the parental Vk gene, however three had Vk genes derived fromthe Vk3 family. The Vk genes from the affinity matured Fab had 16 to 33amino acid substitutions compared to the parental Vk gene (Table 13).

Generation and Characterization of IgG Constructed from the V-Genes ofFab

To determine whether the affinities observed for the Fab wererecapitulated in IgG, and to generate antibodies for the diagnosis andtreatment of botulism, we converted 2G11, B6.1 and B11E8 to humanIgG1/kappa antibodies. IgG were expressed from stable CHO cell lines,purified using protein G and solution K_(D) for BoNT/A or BoNT/Bsubtypes measured by flow fluorimetry. The affinities of the three IgGranged from 25.1 to 6.59 pM for BoNT/A1 or BoNT/B1 and were increasedfrom 4.85 fold to 42 fold compared to the affinities measured for theyeast displayed Fab (Table 14). We have previously observed comparableincreases in affinities for yeast displayed scFv converted to IgG. 2G11bound three BoNT/A subtypes (Bont/A1, A2, and A3 with comparable andhigh affinity. B6.1 bound four BoNT/B subtypes (BoNT/B1, B2, B3, and B4)with comparable and high affinity. In contrast, B11E8 only boundBoNT/B1, B2, and B3 (Table 14).

TABLE 14 Solution equilibrium binding constants for IgG constructed fromaffinity matured BoNT Fab. Equilibrium dissociation constants (K_(D))for affinity matured BoNT IgG were measured for the different BoNTsubtypes by flow fluorimetry in a KinExA. IgG Affinity bv KinExA CloneK_(D) (×10⁻¹²M⁻¹) BoNT/A1 BoNT/A2 BoNT/A3 2G11 25.1 40.4 18 BoNT/B1BoNT/B2 BoNT/B3 BoNT/B4 B6.1 6.82 9.18 28.5 9.41 B11E8 6.59 18.1 15.6 NB

Example 3 Neutralization of BoNT/B In Vivo by BoNT/B AntibodyCombinations

In vivo, as found for BoNT/A, single mAbs protected mice against onlyrelatively low BoNT/B challenge doses (up to 100 mouse LD50s per 50 μgof mAb). But for BoNT/A and BoNT/E, mAb combinations showed much morepotent neutralization than for BoNT/B neutralization. The most potentthree mAb combination (2B18.1:1B10.1:B12.1), however, only protectedmice up to a challenge dose of 40,000 LD₅₀s/50 μg of antibody, about 10fold less than seen for three mAb combinations against BoNT/A and BoNT/E(Table 15). Addition of a fourth mAb (B11E8) increased potency of about50 fold, such that all mice receiving 2 μg total of antibody werecompletely protected against challenge with 40,000 mouse LD₅₀s ofBoNT/B1. Since 1 IU for BoNT/B is the amount of antibody neutralizing10,000 LD₅₀s of BoNT/B (50% of mice survive), this would translate intoa potency of about 2000-4000 IU/mg. Thus a total human therapeutic doseequivalent to the 5500 IU of licensed equine antitoxin used to treattype B botulism would be less than about 2.5 mg. Other combinations offour mAbs had comparable potency against BoNT/B1. Protection againstchallenge by BoNT/B2 by mAbs 2B18.1:1B10.1:B12.1:B11E8 was about 4 to 5fold less than the protection seen for BoNT/B1, similar to the reducedpotency seen against this subtype for the equine antitoxin. The reasonfor the reduced potency is not clear, since the mAbs bind BoNT/B2 withaffinities comparable to the affinities for BoNT/B1. Regardless, thefour mAb combination is still highly potent against BoNT/B2. Based onthe significantly higher potency observed for the four mAb combinations,a combination of four mAbs rather than three may be preferable forBoNT/B antitoxin compositions.

TABLE 15 In vivo potency of BoNT/B neutralization by BoNT/B antibodycombinations Mice surviving per 10 dosed 10,000 20,000 40,000 40,00040,000 40,000 40,000 LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ amount Antibodymixture 50 μg 50 μg 50 μg 10 μg 5 μg 2 μg 1 μg BoNT/B12B18.1:1B10.1:B12.1 10 10 6 0 2B18.1:1B10.1:B12.1:B11E8 10 10 10 32B18.1:B10.1:B12.1:B6.1 10 10 9 1 2B18.1:B6.1:B11E8:B12.1 10 10 7BoNT/B2 2B18.1:1B10.1:B12.1 4 0 2B18.1:1B10.1:B12.1:B11E8 10 10 10 6 0

An equimolar combination of the indicated amount of mAb was mixed withthe indicated BoNT/B subtype and injected into mice. The amount of mAbgiven is the total dose of the four mAb combination. The number of micesurviving of 10 studied is reported.

Example 4 Neutralization of BoNT/E In Vivo by BoNT/E AntibodyCombinations

In vivo, as found for BoNT/A, single mAbs protected mice against onlyrelatively low BoNT/E challenge doses (up to 200 mouse LD₅₀s per 50 μgof mAb). Similar as for BoNT/A, mAb combinations showed highly potentBoNT/E neutralization. In vivo, 5-10 ug of the most potent combinationof three mAbs (3E2, 3E6.1, and 4E16.1) protected mice challenged with40,000 mouse LD₅₀s of BoNT/E1 and BoNT/E3 (Table 16). Since 1 IU ofBoNT/E antibody neutralizes 1,000 mouse LD₅₀s, about 1 mg of this mAbcombination would be the equivalent of 8,000 IU of antitoxin for BoNT/E1or E3. This is equivalent to the human therapeutic dose ofinvestigational BoNT/E equine antitoxin given to treat type E botulism.The potency of some of the different three mAb combinations of the fourdifferent BoNT/E mAbs was also studied, and found to be approximatelyequipotent in the three mAb combination.

TABLE 16 In vivo potency of BoNT/E neutralization by BoNT/E antibodycombinations Mice surviving per 10 dosed 10,000 20,000 40,000 40,00040,000 40,000 40,000 LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ amount Antibodymixture 50 μg 50 μg 50 μg 10 μg 5 μg 2 μg 1 μg BoNT/E1 3E2:3E6.1:4E16.110 10 10 8 10 0 3E2:3E6.1:4E17.1 10 10 0 BoNT/E3 3E2:3E6.1:4E16.1 10 9 90 3E2:3E6.1:4E17.1 10 10 2 3E2:3E16.1:4E17.1 10 10 9 1

An equimolar combination of the indicated amount of three mAbs was mixedwith the indicated BoNT/E subtype and injected into mice. The amount ofmAb given is the total dose of the three mAb combination. The number ofmice surviving of 10 dosed is reported.

Example 5 Residues Involved in the Binding of Various IgG to BoNT

Methods and Materials

Oligonucleotide Primers

Standard PYD2 primers: pair Gap 5 and Gap 3 or pair PYDFor and PYDRevare used for per for sequencing and/or cloning. Gap 5 and Gap3-1 areused for DNA sequencing.

Gap repair 5′ (Gap5):  5′-TTAAGCTTCTGCAGGCTAGTG-3′ (SEQ ID NO: 932)Gap repair 3′ (Gap3):  5′-GAGACCGAGGAGAGGGTTAGG-3′ (SEQ ID NO: 933)Gap repair 3′-1 (Gap 3-1): 5′-GAGAGGGTTAGGGATAGG-3′ (SEQ ID NO: 934)pYDFor:  5′-AGTAACGTTTGTCAGTAATTGC-3′ (SEQ ID NO: 935) PYDRev: 5′-GTCGATTTTGTTACATCTACAC-3′ (SEQ ID NO: 936)

Primers for amplifying and cloning the target antigen gene by gaprepair:

Ag-Gap5 primer  5′GTTGTTCTGCTAGCGGGGCCAATGG--------------------------3′(SEQ ID NO: 937) Ag-Gap3 primer 5′-TTCGAAGGGCCCGCCTGCGGCCGC--------------------------3′ (SEQ ID NO: 490)The 5′ sequence indicated anneals to Nco1-Not1 digested pYD2 DNA forcloning by gap repair, the bolded sequences are the Nco1 and Not1 sites.The dashed sequence should include approximately 24 nucleotides thatanneal to the 5′ and 3′ ends of the target antigen DNA. Primers foramplifying and introducing alanine on the target antigen gene: Theseprimers are designed specifically for each toxin domain and mutation toconstruct

Strains, Media, and Reagents

Pfu DNA Polymerase (Stratagene); Taq Polymerase (New England Biolabs(NEB)); GENECLEAN TURBO kit (MP Biomedicals, LLC, Cat. 1102-600); CustomDNA primers (many vendors); deoxynucleotide triphosphates (NEB); Nco I,Not I restriction enzymes (NEB); SV5 Antibody (Invitrogen); APCconjugated Fab-specific goat-anti-human (Fab)₂ (Jackson ImmunoResearch); Fab preparation kit (Pierce product number 44985);saccharomyces cerevisiae EBY100 (Invitrogen); 1 M lithium acetate(LiAc); 0.1 M LiAc; 2 mg/mL single stranded DNA (ssDNA); polyethyleneglycol (PEG) 3350 (50%); plasmid vector pYD2 as describe above; YPDmedium (recipe: to 900 mL deionized H₂O add: 10 g Yeast extract and 20 gPeptone. Optional: Add 17 g agar/L for plates. autoclave, cool to 55-60°C. Add 100 ml of 20% dextrose (filter sterilized). For liquid mediapreparation filter sterilize (0.22 um filter) after all components havebeen added and dissolved.) SD-CAA medium (recipe: to 900 mL deionizedH₂O add: 7 g Yeast Nitrogen base w/o amino acid, 10.19 g Na₂HPO₄.7H₂O or5.4 g Na₂HPO₄, 8.56 g NaH₂PO₄.H₂O or 7.4 g NaH₂PO₄, and 5 g CAA (DIFCO)w/o Tryptophan or Ura. After all components dissolve, add 100 ml of 20%dextrose and 10 ml of 0.6% (100×) Leucine. Sterilize by filteringthrough 0.22 μm filter; SG-CAA medium (recipe: to 900 mL deionized H₂Oadd: 7 g Yeast Nitrogen base w/o amino acid, 10.19 g Na₂HPO₄.7H₂O or 5.4g Na₂HPO₄, 8.56 g NaH₂PO₄.H₂O or 7.4 g NaH₂PO₄, and 5 g CAA (DIFCO) w/oTryptophan or Ura. After all components dissolve, add 100 ml of 20%galactose and 10 ml of 0.6% (100×) Leucine. Sterilize by filteringthrough 0.22 μm filter; ALEXA FLUOR 647 dye labeling kit (MOLECULARPROBES); ALEXA FLUOR 488 dye labeling kit (MOLECULAR PROBES) conjugatedanti-human Fc specific antibody (Jackson Immunoresearch); FACS Buffer:to 1 L 1×PBS add 5 g BSA (final 0.5%), 1 mL 1M MgCl₂ (final 1 mM) and0.5 mL 1 M CaCl₂ (final 0.5 mM), sterile filter; FACS-ARIA Cell Sorter(BD Biosciences); Pure BoNToxins (USAMIIRID, Metabiologics); recombinantBoNToxins Fragments (UASAMIIRID).

Specificity and Crossreactivity Test

5.0×10⁵ scFv/yeast of an induced culture, from the scFv to test, wereincubated with 100 uL of a 10 nM solution of the toxin to test. After 2hours of incubation at 4° C., cells were washed with 500 uL of cold FACSbuffer and resuspended in 200 uL of the detecting antibodies. Binding ofBoNToxins to yeast-displayed scFv was detected using a 1:500 dilution of1 mg/ml mAb binding a non-overlapping epitope labeled with ALEXA FLUOR647 dye. Expression of yeast cells was detected using and anti-SV5 tagIgG labeled with ALEXA FLUOR 488 dye, 1:300 dilution.

This test was repeated for each available subtype of the correspondingBoNToxin. To test for nonspecific binding, cells were also incubatedwith the detection antibodies only. No toxin was used for this test.Recognition of each toxin was estimated by measuring yeast fluorescencein a FACS ARIA cell sorter, using the FITC and APC channels.

Measurement of Yeast-Displayed scFv Affinity for the CorrespondingBoNToxin

Quantitative equilibrium binding was determined using yeast-displayedscFv and flow cytometry as described previously (Boder et al. Proc.Natl. Acad. Sci. USA 2000, 97:10701-10705) In general, six to eightdifferent concentrations of pure BoNT/A were used spanning a range ofconcentrations from ten times above to ten times below the KD.Incubation volumes and number of yeast stained were chosen to keep thenumber of antigen molecules in fivefold excess above the number of scFv,assuming 5.0×10⁵ scFv/yeast. Incubation times were chosen based onanticipated times to equilibrium calculated using approximations of theanticipated association rate constant (kon) and dissociation rateconstant (k_(off)). For the higher affinity scFv, this was as long as18-24 h. Binding of BoNToxins to yeast-displayed scFv was detected asfor specificity test. Each KD was determined in triplicate, threeseparate inductions and measurements. To measure the antibody-toxinaffinity constant (K_(D)) within the surface display context, only thescFv displaying yeast (SV5 binding) were included in the analysis byco-staining with SV5-Alexa-488. Affinity determination was performed foreach one of the toxin subtypes recognized by the scFv being analyzed.

Binding Domain Test

Binding to recombinant purified toxin domains was performed using thesame protocol as for the whole toxin, on the yeast-displayed scFv. Whenrecombinant toxin was not available the desired toxin domains werecloned and displayed in EBY 100 yeast cells (Levy et al, supra). Onlythe IgG format, or free scFv can be used in this test. Binding to theyeast displayed toxin domain was performed by incubating 5.0×10⁵ inducedcells with 100 uL of the IgG-ALEXA FLUOR 647 dye to test. 1:500 dilutionof the labeled IgG plus 1:300 dilution of SV5-488 were used. Cells wereincubated at 4° C. for 30 min and after washing with cold FACS buffer,fluorescence was measured.

Construction of Yeast-Displayed BoNToxins Fragments, Wild Type orAlanine Mutants.

Genes encoding the desired botulinum neurotoxin fragments (Lc, Hn or HC)in pYD2, wt fragments or alanine mutated, were constructed by using PCRand gap repair. Primers For and Rev were designed to amplify thesynthetic gene fragment, adding the restriction sites NcoI and NotI tothe 5′ and 3′ ends, respectively. The pYD2 vector was digested with NcoIand Nod, and together with the gel purified per fragment used totransform EBY100 by gap repair (Orr-Weaver et al. Proc Natl Acad Sci USA1983, 80: 4417-21; Gietz et al. Yeast 1983, 7: 253-63). Clonescontaining the correct insert were confirmed by DNA sequencing to yieldpYD2/Toxin fragment.

For each Alanine mutant constructed a pair of specific oligos weredesigned and used in combination with the pYD2 standard oligos pYDForand pYDRev to construct the mutated fragment by per. Transformation wasperformed, by gap repair, using the double digested plasmid and the twoper products, all gel purified. After induction, mutated fragments haddisplay levels resulting in at least a 1.5 log shift when stained withSV5-ALEXA FLUOR 488 dye, comparable to the levels of wild-type fragment.

Generation of IgG from scFv

Each scFv of interest was converted into full length IgG. Briefly, genesencoding scFv VH and VL were amplified using PCR from their respectivepYD2 vectors with a designed primer pair and cloned into plasmid N5KG1.Clones containing the correct genes were identified by DNA sequencingand vector DNA was used to transfect CHO DG44 cells by electroporation.Stable cell lines were established by selection in G418 and expandedinto 1-liter spinner flasks. Supernatant containing IgG was collected,concentrated by ultrafiltration and purified on Protein G (Pharmacia).IgG purity was assessed by native and denaturing SDS-PAGE andconcentration determined by absorbance at 280 nm.

Epitope Sharing Test

Each scFv to test was stained with the corresponding toxin, as describedabove for the specificity test. Detection was done, separately, by eachexisting IgGs that binds to the toxin used. Fluorescence measurementswere used as indicator of the spatial proximity of both epitopes, forthe scFv and for the IgG. No signal meant that epitopes were too closeand both antibodies could not bind simultaneously. The contrary wasassumed for a strong signal, epitopes were well separated permittingbinding of both antibodies.

After full characterization of each scFv an analysis of the toxinsequences was performed but only for the determined binding fragment.For an antibody that had been shown to bind the Hn domain, ortranslocation domain, toxin alignment was done only for the sequencesregions corresponding to the Hn domains of the toxin subtypes.

Protein Sequence Alignment

The protein analysis and multiple sequence alignment were performedusing the ClustalW application from the Mac Vector Software (Mac Vector,INC.). All known sequences for the different subtypes of the BoNTserotype were used to find the areas that presented antigen differences.The differential binding to each subtype was used as an indicator of aprobable binding site, normally several regions were found. Each regionwas located in the 3D structure of the whole toxin assessing the levelof exposure and conservation of the amino acids in the area.

The aim was to locate areas that could explain the binding pattern ofthe antibody and then test by alanine scanning the energetic importanceof each site. Mutated amino acids that led to loss of binding wereconsidered as energetically favorable contacts. Further single yeastdisplayed alanine mutants of the putative epitope site are constructedand antibody binding determined in order to identify the epitope.

Measurement of the Affinity of Fab Fragments for Yeast Displayed WildType and Alanine Mutants Toxin Domains.

By measuring the affinity of antibody for the alanine mutants comparedto the wild type yeast displayed antigen, it is possible to determinethe energetic contribution of the amino acid side chain to antibodybinding.

The dissociation equilibrium constants (K_(D)) of the Fab fragment ofthe antibody to test for wild type and alanine mutants of yeastdisplayed toxin fragments were measured by flow cytometry on a FACS ARIAcell sorter. First, EBY100 yeast cultures harboring the pYD2/toxindomain, wild type or the pYD2/alanine mutant plasmids were grown andinduced as described above for yeast-displayed scFv. Aliquots of 1×10⁵induced yeast cells (0.005 OD600 ml⁻¹) were washed in FACS buffer andincubated with dilutions (according to the IgG K_(D)) of the Fabfragments such that the K_(D) would be spanned by at least fivefold,where possible. Incubation volumes were chosen to ensure that a tenfoldmolar excess of the antibody (ligand) over the displayed moiety (H_(C),H_(N) or L_(C)) would be maintained. For this purpose, it was assumedthat 10⁵ copies of the toxin domain were displayed on the yeast surface.Incubation with the Fab was allowed to proceed for 4 h at 23° C. Cellswere then washed in FACS buffer and resuspended inallophycocyanin-conjugated Fab-specific goat-anti-human F(ab)¢2 at1:1000 dilution in FACS buffer. To accurately determine the K_(D) of theFab fragments within the surface display context, we included onlyprotein displaying yeast in the analysis by staining with SV5 (ALEXAFLUOR 488 dye Alex-ft-48-8) mAb (VanAntwerp et al. Biotechnol. Prog.2000, 16:31-37).

Calculation of the Change in Free Energy of Binding to the Wild Type andAlanine Mutants.

For each alanine mutation, the change of free energy (DDGmut-wt) betweenthe alanine (Ala) mutant relative to that of the wild type (wt) wascalculated using the following standard formula and using the previouslymeasured KD constants:ΔΔG _(mut-wt) =RT ln(K _(Dmut) /K _(Dwt))

These calculations provide a measure of the energetic contribution ofeach one of the alanine substituted amino acid residues, for the mAb,therefore indicating the position of its functional epitope. Fab wasused as it is monovalent, compared to the bivalent IgG. This eliminatesthe avidity effect that can occur with bivalent binding which can leadto falsely high (and inaccurate) affinities (Levy et al, supra).

TABLE 17 Epitope Data IgG Toxin/Domain Residues Involved 4E17.1 A1/HnY753 ,E756, E757 1B18 A1/Hn Y750, N751, T754 HuC25 A1/Hc E920, F953,H1064 AR2/CR1 A1/Hc B918, L919, E920, F953, D1062, T1063, H10643D12/RAZ1 A1/Hc G1129, I1130, R1131 1B11 B1/Hn I549 , S565 4E17.1 B1/HnK747, R749 , Y750, N751 1B18 B1/Hn N751, Y750, Y753 3E1 E1/Lc N14 , D15, R16, Q29, E30, Y32 , E135 , K137 , F138 , N140 , S142, Q143 , D144 ,I145 3E3 E1/Lc N14 , Y32, E135 , K137 , F138 , N140, S142, Q143 , D144 ,I145 3E5 E1/Lc N14 , D15, Y30 , D144 3E6.1 E1/Lc S604 , Q607 , Q608 4E11E1/Lc N14 , D15, E30, Y32 4E16.1 E1/Lc N14 , D15 , E30, Y32 , D1444E17.1 E1/Hc E754b , E755

The numbering system used for the residues of toxin in the table aboveis based on Lacy et al. (1999) J. Mol. Biol. 291:1091-1104. Residuesthat are bolded and underlined were found to have importantcontributions energetically and eliminations of the residues often leadto total loss of binding the corresponding antibodies.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

While the subject antibody, method, and composition have beenparticularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims.

What is claimed is:
 1. An isolated antibody that competes for binding toa Botulinum neurotoxin with an antibody comprising: a V_(H) CDR1comprising the amino acid sequence NYPMS (SEQ ID NO:520); a V_(H) CDR2comprising the amino acid sequence SLTASGDNTFYADSVKG (SEQ ID NO:522); aV_(H) CDR3 comprising the amino acid sequence of ALVGRYDISTGYYRPVMDS(SEQ ID NO:524); a V_(L) CDR1 comprising the amino acid sequence ofRTSQGFTSALA (SEQ ID NO:770); a V_(L) CDR2 comprising the amino acidsequence of DASKLES (SEQ ID NO:772); and a V_(L) CDR3 comprising theamino acid sequence of QQSNSYPLT (SEQ ID NO:774).
 2. The isolatedantibody of claim 1, wherein the antibody specifically binds an epitopeof a Botulinum neurotoxin that is specifically bound by an antibodycomprising: a V_(H) CDR1 comprising the amino acid sequence NYPMS (SEQID NO:520); a V_(H) CDR2 comprising the amino acid sequenceSLTASGDNTFYADSVKG (SEQ ID NO:522); a V_(H) CDR3 comprising the aminoacid sequence ALVGRYDISTGYYRPVMDS (SEQ ID NO:524); a V_(L) CDR1comprising the amino acid sequence RTSQGFTSALA (SEQ ID NO:770); a V_(L)CDR2 comprising the amino acid sequence DASKLES (SEQ ID NO:772); and aV_(L) CDR3 comprising the amino acid sequence QQSNSYPLT (SEQ ID NO:774).3. The isolated antibody of claim 1, wherein the antibody comprises: avariable heavy chain (V_(H)) comprising the amino acid sequence of SEQID NO:23; and a variable light chain (V_(L)) comprising: a V_(L) CDR1comprising the amino acid sequence RTSQGFTSALA (SEQ ID NO:770); a V_(L)CDR2 comprising the amino acid sequence DASKLES (SEQ ID NO:772); and aV_(L) CDR3 comprising the amino acid sequence QQSNSYPLT (SEQ ID NO:774).4. The isolated antibody of claim 3, wherein the V_(L) comprises theamino acid sequence of SEQ ID NO:59.
 5. The isolated antibody of claim1, wherein the antibody comprises: a variable heavy chain (V_(H))comprising: a V_(H) CDR1 comprising the amino acid sequence NYPMS (SEQID NO:520); a V_(H) CDR2 comprising the amino acid sequenceSLTASGDNTFYADSVKG (SEQ ID NO:522); and a V_(H) CDR3 comprising the aminoacid sequence ALVGRYDISTGYYRPVMDS (SEQ ID NO:524); and a variable lightchain (V_(L)) comprising the amino acid sequence of SEQ ID NO:59.
 6. Theisolated antibody of claim 5, wherein the V_(H) comprises the amino acidsequence of SEQ ID NO:23.
 7. The isolated antibody of claim 2, whereinthe antibody comprises: a variable heavy chain (V_(H)) comprising theamino acid sequence of SEQ ID NO:23; and a variable light chain (V_(L))comprising: a V_(L) CDR1 comprising the amino acid sequence RTSQGFTSALA(SEQ ID NO:770); a V_(L) CDR2 comprising the amino acid sequence DASKLES(SEQ ID NO:772); and a V_(L) CDR3 comprising the amino acid sequenceQQSNSYPLT (SEQ ID NO:774).
 8. The isolated antibody of claim 7, whereinthe V_(L) comprises the amino acid sequence of SEQ ID NO:59.
 9. Theisolated antibody of claim 2, wherein the antibody comprises: a variableheavy chain (V_(H)) comprising: a V_(H) CDR1 comprising the amino acidsequence NYPMS (SEQ ID NO:520); a V_(H) CDR2 comprising the amino acidsequence SLTASGDNTFYADSVKG (SEQ ID NO:522); and a V_(H) CDR3 comprisingthe amino acid sequence ALVGRYDISTGYYRPVMDS (SEQ ID NO:524); and avariable light chain (V_(L)) comprising the amino acid sequence of SEQID NO:59.
 10. The isolated antibody of claim 9, wherein the V_(H)comprises the amino acid sequence of SEQ ID NO:23.
 11. The isolatedantibody of claim 1, wherein the antibody is humanized.
 12. The isolatedantibody of claim 1, wherein the antibody is selected from the groupconsisting of: a single chain Fv (scFv), an IgG, a Fab, a (Fab′)₂, andan (scFv′)₂.
 13. The isolated antibody of claim 1, wherein the antibodyis an IgG.
 14. The isolated antibody of claim 2, wherein the antibody ishumanized.
 15. The isolated antibody of claim 2, wherein the antibody isselected from the group consisting of: a single chain Fv (scFv), an IgG,a Fab, a (Fab′)₂, and an (scFv′)₂.
 16. The isolated antibody of claim 2,wherein the antibody is an IgG.
 17. A conjugate comprising: the antibodyof claim 1; and a label.
 18. The conjugate of claim 17, wherein thelabel is selected from the group consisting of: a fluorescent label, aspectroscopic label, an enzyme, a colorimetric label, a chemiluminescentlabel, biotin, and streptavidin.
 19. The conjugate of claim 17, whereinthe antibody specifically binds an epitope of a Botulinum neurotoxinthat is specifically bound by an antibody comprising: a V_(H) CDR1comprising the amino acid sequence NYPMS (SEQ ID NO:520); a V_(H) CDR2comprising the amino acid sequence SLTASGDNTFYADSVKG (SEQ ID NO:522); aV_(H) CDR3 comprising the amino acid sequence of ALVGRYDISTGYYRPVMDS(SEQ ID NO:524); a V_(L) CDR1 comprising the amino acid sequence ofRTSQGFTSALA (SEQ ID NO:770); a V_(L) CDR2 comprising the amino acidsequence of DASKLES (SEQ ID NO:772); and a V_(L) CDR3 comprising theamino acid sequence of QQSNSYPLT (SEQ ID NO:774).
 20. A kit comprisingthe conjugate of claim
 17. 21. A pharmaceutical composition comprising:the antibody of claim 1; and a pharmaceutically acceptable carrier. 22.The pharmaceutical composition of claim 21, wherein the antibodyspecifically binds an epitope of a Botulinum neurotoxin that isspecifically bound by an antibody comprising: a V_(H) CDR1 comprisingthe amino acid sequence of SEQ ID NO:520; a V_(H) CDR2 comprising theamino acid sequence of SEQ ID NO:522; a V_(H) CDR3 comprising the aminoacid sequence of SEQ ID NO:524; a V_(L) CDR1 comprising the amino acidsequence of SEQ ID NO:770; a V_(L) CDR2 comprising the amino acidsequence of SEQ ID NO:772; and a V_(L) CDR3 comprising the amino acidsequence of SEQ ID NO:774.
 23. A kit comprising the pharmaceuticalcomposition of claim
 21. 24. A method of specifically binding aBotulinum neurotoxin in a mammal comprising: administering to the mammalan effective amount of the antibody of claim 1, wherein theadministering provides for binding of the antibody to the botulinumneurotoxin present in the mammal.
 25. The method according to claim 24,wherein the antibody specifically binds an epitope of a Botulinumneurotoxin that is specifically bound by an antibody comprising: a V_(H)CDR1 comprising the amino acid sequence NYPMS (SEQ ID NO:520); a V_(H)CDR2 comprising the amino acid sequence SLTASGDNTFYADSVKG (SEQ IDNO:522); a V_(H) CDR3 comprising the amino acid sequence ofALVGRYDISTGYYRPVMDS (SEQ ID NO:524); a V_(L) CDR1 comprising the aminoacid sequence of RTSQGFTSALA (SEQ ID NO:770); a V_(L) CDR2 comprisingthe amino acid sequence of DASKLES (SEQ ID NO:772); and a V_(L) CDR3comprising the amino acid sequence of QQSNSYPLT (SEQ ID NO:774).
 26. Amethod of determining the presence or absence of specifically binding aBotulinum neurotoxin in a biological sample, comprising: contacting thebiological sample with the conjugate of claim
 17. 27. The methodaccording to claim 26, wherein the antibody specifically binds anepitope of a Botulinum neurotoxin that is specifically bound by anantibody comprising: a V_(H) CDR1 comprising the amino acid sequenceNYPMS (SEQ ID NO:520); a V_(H) CDR2 comprising the amino acid sequenceSLTASGDNTFYADSVKG (SEQ ID NO:522); a V_(H) CDR3 comprising the aminoacid sequence of ALVGRYDISTGYYRPVMDS (SEQ ID NO:524); a V_(L) CDR1comprising the amino acid sequence of RTSQGFTSALA (SEQ ID NO:770); aV_(L) CDR2 comprising the amino acid sequence of DASKLES (SEQ IDNO:772); and a V_(L) CDR3 comprising the amino acid sequence ofQQSNSYPLT (SEQ ID NO:774).
 28. A method of specifically binding aBotulinum neurotoxin in a mammal, comprising: administering to themammal an effective amount of the pharmaceutical composition of claim21; wherein the administering provides for binding of the antibody tothe botulinum neurotoxin present in the mammal.
 29. The method accordingto claim 28, wherein the antibody specifically binds an epitope of aBotulinum neurotoxin that is specifically bound by an antibodycomprising: a V_(H) CDR1 comprising the amino acid sequence NYPMS (SEQID NO:520); a V_(H) CDR2 comprising the amino acid sequenceSLTASGDNTFYADSVKG (SEQ ID NO:522); a V_(H) CDR3 comprising the aminoacid sequence of ALVGRYDISTGYYRPVMDS (SEQ ID NO:524); a V_(L) CDR1comprising the amino acid sequence of RTSQGFTSALA (SEQ ID NO:770); aV_(L) CDR2 comprising the amino acid sequence of DASKLES (SEQ IDNO:772); and a V_(L) CDR3 comprising the amino acid sequence ofQQSNSYPLT (SEQ ID NO:774).